An in vitro investigation of the impact of PAX6 haploinsufficiency on human corneal stromal cell phenotype Carla Sanchez Martinez Supervisor: Professor Julie T Daniels Institute of Ophthalmology University College London Thesis submitted to UCL for the degree of Doctor of Philosophy September 2019 1 2 ‘I, Carla Sanchez Martinez confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis.' 3 4 ABSTRACT Aniridia related keratopathy (ARK), caused by PAX6 haploinsufficiency, leads to corneal opacification and sight loss. ARK is still an unmet clinical need, as treatments are not always available or successful. Therefore, it is essential to clarify the biological mechanisms behind ARK to develop new efficient clinical treatments. It was hypothesized that aniridic corneal stromal cells are affected and contribute to the development of ARK. To test this hypothesis, stromal cells were isolated from central human aniridic corneas and compared to normal corneal stromal cells isolated from healthy donors. Cells were cultured in 2D and in 3D tissue equivalent culture models to identify ARK-associated features. For the first time, aniridic and normal corneal stromal cells were successfully cultured and expanded in vitro. Unexpectedly, aniridic and normal corneal stromal cells showed similar phenotype in 2D. They presented similar stem cell-like characteristics when cultured in 2% FBS containing media and showed the potential to differentiate into keratocyte-like cells when differentiated in serum-free medium. Aniridic corneal stromal cells showed reduced proliferation capacity when cultured both in 2D and in 3D. Moreover, when cultured inside the 3D tissue equivalent, aniridic corneal stromal cells showed different morphology and distinct spatial distribution when compared to normal cells. RNA sequencing data showed major differences between aniridic and normal corneal stromal cells transcriptomes (with 153 differentially expressed genes being identified), with significant differences in expression levels of genes involved in several biological processes, including matrix remodelling. Real-Time qPCR and ELISA confirmed lower expression and secretion levels of MMP1 and MMP2 (proteins involved on matrix remodelling) by aniridic corneal stromal cells when compared to their normal counterparts. In conclusion, it was demonstrated for the first time that aniridic corneal stromal cells can be successfully cultured in 2D and 3D environments. The differences observed between aniridic and normal corneal stromal cell phenotypes in vitro, suggest that aniridic corneal stromal cells have impaired functionality in vivo and therefore might contribute to the development of ARK. 5 6 IMPACT STATEMENT The cornea is our window to the world and its transparency is essential for vision. Corneal transparency can be compromised by several congenital disorders, including aniridia, that is caused by mutations in the PAX6 gene and affects different parts of the eye. The most severe consequence of aniridia is the so-called aniridia related keratopathy (ARK). ARK is a rare condition that starts to affect individuals from their teenage years and can progress, leading to corneal opacification and sight loss. For these patients, ARK represents a significant clinical problem, as effective treatments are not always available. Superficial keratectomy, penetrating keratoplasty and experimental cultured LESC therapy may be used for the treatment of ARK corneas with high levels of opacification. However, there are different reasons why these treatments are not always available or successful: there is a shortage of corneal donors for transplantation worldwide and aniridic patients present variable responses and high levels of scarring following surgery. In order to develop new therapeutic approaches, it is of crucial importance to better understand the biological mechanisms driving ARK. For the first time, we showed that human ARK-derived corneal stromal cells can be successfully cultured and expanded in vitro, and that they present phenotypical differences to normal cells. These results are the platform for future studies to carry on the in vitro characterisation of this condition. Moreover, the results presented in this thesis provide further understanding on the mechanisms behind ARK. We provided additional evidence that suggests a stromal involvement during the progression of ARK, which adds to the current body of research on this condition. Our results not only have an impact in academia but will also be of utmost interest for the patient community. The hypothesis that ARK derived corneal stromal stem cells might contribute to the development of ARK, opens a new window for using them as a novel therapeutic target. In a tangible example, an ongoing clinical trial in our laboratory aims to replace both the epithelium and the stroma in ARK patients with healthy cells cultured in a tissue engineering approach. This is a great advancement, and the results here presented provide evidence supporting the success of this reconstructive therapy. Lastly, it is noteworthy that aniridia is a rare disease with a motivated and engaging patient network. We recently presented this work to the patient community, who are eager on updates on the most recent advances in aniridia research. In the near future, this work might also be used in platforms such as Aniridia Network UK to raise awareness for this condition, and to bridge the knowledge gap between the research community and the public. 7 In conclusion, our research will impact the research community in the short term, by going a step forward to the understanding of the mechanisms behind ARK. In the long term, it will hopefully have a clinical use, by providing the ground work to develop new therapeutic strategies. 8 ACKNOWLEDGEMENTS The completion of the thesis here presented has been possible thanks to the support from a wide range of people, to whom I will be forever grateful. To all the patients (and their families) who kindly donated their tissue, without whom this project would not have been possible, thank you. To Fundació La Caixa and Moorfields Eye Charity, for their financial support. I would like to start by expressing my sincere thanks to my supervisor, Professor Julie Daniels, for her guidance and encouragement during the course of this project and for her help in correcting this thesis. I am also thankful to Dr. Victoria Tovell for introducing me to the aniridia project and for teaching me numerous techniques. I would also like to thank my secondary supervisor, Prof. Mike Cheetham, for his guidance during the upgrade process. Finally, I would like to thank Dr. Cerys Evans for her invaluable help during the genotyping of the aniridic samples. I am extremely grateful to my lab colleagues for their help, support and friendship. I will miss those Friday morning coffees with the rounds of: “Anything fun happening this weekend?”. A special shout out goes to my portuguese twin, Rita Pinho, for being the best cell factory buddy and the greatest friend. I would also like to especially thank Dr. Tiago Ramos for being always super energetic and helping me in any possible way. To my friends from the 3rd floor, also known as Tercer piso guay, you have made everything better. We have shared great experiences not only at IoO but also in Hawaii, Canada and Barcelona. I really hope that the list keeps getting longer because I am going to miss you all. I also wish to thank my friends for their support during these four years, with a special mention to (soon to be Dr.) Roser Cañigueral for sharing the writing process during these past few weeks in our home away from home. I would also like to especially mention Dr. Javier Uceda, whose smile was and still is the greatest inspiration. His memory is a constant reminder to take joy in every single aspect of our lives. Finally, this would not have been possible without the support from my family, the beques group, and especially my parents Anna Martinez and Eduard Sanchez. They have taken the best care of me, making sure I go back home often and never failing to ask when I would be coming back before I have even left. Gràcies per fer-me costat durant aquest viatge i per preocupar-vos de mi i de les meves cèl·lules. Thank you, 9 10 TABLE OF CONTENTS Abstract ....................................................................................................................... 5 Impact statement ........................................................................................................ 7 Acknowledgements .................................................................................................... 9 Table of contents ...................................................................................................... 11 List of figures ............................................................................................................ 17 List of tables ............................................................................................................. 21 Abbreviations ............................................................................................................ 23 CHAPTER 1: Introduction ................................................................................. 25 1.1 The ocular surface: The cornea .................................................................... 25 Overview ..............................................................................................