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In Vitro Generation of Human Retinal Ganglion Cells Via Direct Conversion and Stem Cell Differentiation

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In Vitro Generation of Human Retinal Ganglion Cells Via Direct Conversion and Stem Cell Differentiation IN VITRO GENERATION OF HUMAN RETINAL GANGLION CELLS VIA DIRECT CONVERSION AND STEM CELL DIFFERENTIATION by Valentin Mikhaylovich Sluch A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland November, 2015 © 2015 Valentin Mikhaylovich Sluch All Rights Reserved Abstract Retinal ganglion cells (RGCs) are essential for human visual perception, as they mediate the transfer of visual information from the eye to the brain. In the event of RGC death, as happens in diseases such as glaucoma, vision is permanently lost because the mammalian central nervous system (CNS) does not regenerate. In order to find novel treatments for the RGC diseases, a number of different research approaches have been undertaken. One relatively recent approach involves the use of pluripotent stem cell (PSC) technologies. As human PSCs can be differentiated to the RGC lineage, it has become possible to utilize human RGCs for drug discovery, disease modeling, or transplantation experiments to attempt visual system recovery. The field of PSC differentiation to RGCs has been steadily developing, but many hurdles remain. Initial reports involved co-culture of mouse stem cells with primary embryonic retinal cells or conditioned media, and yielded only small numbers of cells. Evolution of ocular stem cell biology over the past few years has greatly improved the technology for generation of three-dimensional optic vesicles that develop all of the retinal layers, including the RGC layer. Although such reports were very encouraging, these studies failed to demonstrate that RGCs could be isolated from culture and deeper profiles of the RGCs were missing. Here, we describe a novel, simplified protocol for RGC differentiation from human PSCs. We took advantage of recently developed CRISPR-Cas9 technologies to genetically engineer a stem cell reporter line for RGCs based on the BRN3B/POU4F2 gene. Using this line, we developed a fluorescence- activated cell sorting (FACS) protocol that yields highly purified RGCs, and we ii subsequently characterized the purified cells in terms of their expression pattern of RGC- associated genes and other cellular properties. Despite the power of FACS to provide purified populations of fluorescent RGCs, FACS-based purification schemes also have their limitations. A main limitation is the relatively slow speed and restricted throughput of FACS. In order to get around these limitations, we developed an alternative approach by genetically engineering a unique cell surface antigen driven by the BRN3B gene into stem cells, allowing us to isolate populations of pure differentiated RGCs by affinity purification in an efficient, large scale, and time saving manner that makes possible the use of human RGCs in a variety of studies including high throughput drug discovery screens. In addition to developing this new RGC purification strategy, which has implications for developing improved systems for the purification of a wide variety of different cell types, we have also optimized our initial differentiation protocol by manipulating known signaling pathways via small molecule supplementation to guide the cells toward a retinal cell fate. In addition to developing and characterizing improved stem cell-based approaches for generating RGCs, we have also been pursuing the generation of RGCs from non-stem cell lines through trans-differentiation and direct reprogramming. Through this work, we have identified a combination of four transcription factors that can directly reprogram the human retinal pigment epithelium (RPE) cell line ARPE19 into RGC-like cells. Expression of ATOH7, BRN2, BRN3B, and MYT1 in ARPE19 cells transformed their morphology to a neuronal phenotype and induced the expression of pan-neuronal and RGC-associated genes. Moreover, when overexpressed in stem cells undergoing retinal differentiation, these factors were able to boost the percentage of cells differentiating to iii the RGC lineage, an effect that could be further increased by our previously identified small molecules. Lastly, we discuss the development of a new method to enhance homology directed repair for the purpose of generating reporter lines in an easier and more routine way. Taken together, these studies provide powerful tools for the use of stem cell technology to study RGC differentiation and biology, and the mechanisms of RGC injury and cell death. Additionally, they provide the means to provide well-characterized and large supplies of RGCs for drug discovery screens and lay the groundwork for possible future cell-based therapeutic approaches for treatment of vision loss and blindness from glaucoma and other forms of optic nerve disease. iv Dissertation Referees Graduate Advisor: Donald J. Zack, M.D., Ph.D. Professor, Departments of Ophthalmology, Molecular Biology and Genetics, and Neuroscience, and Institute of Genetic Medicine Dissertation Reader: Nicholas Marsh-Armstrong, Ph.D. Associate Professor, Department of Neuroscience v Acknowledgements I would like to thank my undergraduate advisor, Dr. Michael Massiah, for encouraging me to attend his alma mater of Johns Hopkins University School of Medicine. Dr. Massiah was instrumental in nurturing my scientific curiosity early on and inspiring me to work hard, think analytically, love what you do, and set high expectations. I am extremely grateful to my graduate advisor, Dr. Donald Zack, for providing me with immense guidance and support during my professional and personal development. Don has inspired me to never stop learning and to keep challenging myself as a scientist. He has been a shining example of being the best person you can be and his kindness to others as well as his infectious curiosity have always stood out to me. Don has encouraged all of us to explore and to follow our passions and to never assume something to be impossible because as all Zack lab members know, “it’s always easy enough to try.” It has been an absolute pleasure to work with everyone in the Zack lab, but I am especially indebted to Drs. Julien Maruotti and Karl Wahlin for helping me get started in the fine arts of stem cell culture and molecular biology. I also owe a great deal to Drs. Cindy Berlinicke and Gillian Shaw for their advice and companionship and without whom, grad school would have been a far worse experience. I would like to thank my thesis committee members of Drs. Guo-Li Ming, Valina Dawson, and Nicholas Marsh-Armstrong. I would like to further extend my gratitude to Nick for his mentorship, advice, and collaboration through the years. Nick took me in as vi a clueless summer rotation student, and helped to mold me into a much better scientist. I would also like to recognize some of our many collaborators without whom we would not be able to achieve our experimental goals: Dr. Jeffrey Diamond, Dr. Justin Kerr, Dr. Hai- Quan Mao, Kellin Krick, and Russ Martin. I especially thank Dr. Mao for his enthusiasm and for his help in my career. I would like to thank all of my dear friends and colleagues from Johns Hopkins, especially Chung-ha Davis, Xitiz Chamling, and Judy Nguyen for their invaluable support and insights into life as well as science. If you are the company you keep, then I must say that I have been very fortunate to find myself in fantastic company. I am very grateful to the BCMB program and am highly appreciative for the education I have received here. I also thank my family. Coming out of college, I thought I would pursue structural biology because that had been my prior research discipline. However, my grandfather’s diagnosis and battle with glaucoma pushed me to pursue vision research. Without the support of my parents, Mikhail Sluch and Yulia Soloviova, and my grandparents, I doubt that I would be able to persevere over the many obstacles that I have faced. Lastly, I want to thank my wonderful girlfriend Tonya Gilbert for her love and support. Tonya and I met as fellow BCMB students and have helped each other tremendously throughout our time here. She has been my guiding light in both good times and bad, and our love for each other has helped us to conquer many goals and to grow as people. As our BCMB chapter closes, I very much look forward to our next chapter together. vii Table of contents Abstract……………………………………...........……………….………….…....…..ii-iv Dissertation Referees…………………………...............................................……............v Acknowledgements..........................................................................................................v,vi Chapter 1: General Introduction.....................................................................................1 1.1 The retina and glaucoma................................................................................................1 1.2 Stem cells ………..........................................................................................................2 1.3 RGC genetics and development…………………………………………....…….........7 1.4 Stem cell differentiation to RGCs………………………………………………........11 1.5 RGC transplantation hurdles and possibilities……………………….……………....16 Chapter 2: Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.…….......…..…………...........…….....….…......…......….22 2.1 Introduction……………………………………………………………..…….…….22 2.2 Results……………………………………………………………………....….……24 2.2A Development of an optimized protocol for differentiation of RGCs using an engineered hESC reporter cell line…………………………...……………………....….24
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