Investigating the Role of BEST1 Protein in Best Vitelliform Macular
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Investigating the role of BEST1 protein in Best vitelliform macular dystrophy using a novel, disease-specific human embryonic stem cell line Melissa Maree Mangala BSc (Hons) This thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Medicine, Western Sydney University, Australia 2016 Statement of authentication I hereby, declare that this thesis contains no material that has been accepted for the award of any other degree or diploma and that, to the best of my knowledge and belief, this thesis contains no material previously published or written by another person, except where due reference has been made in the text of this thesis. ___________________ Melissa Maree Mangala Acknowledgements This project was supported by an Australian Postgraduate Award and Western Sydney University top-up scholarship. First and foremost I would like to thank my supervisor Dr. Michael O’Connor; for being a great mentor. Thank you for having faith in me, and investing the time, effort, support, enthusiasm and most importantly patience throughout my candidature. I want to thank my co-supervisors Professor John Morley and Dr. Morven Cameron for their time and support during my project. Thank you for providing the equipment I needed for my electrophysiology experiments, as well as your guidance and knowledge. I would like to give a big shout out to Seaky for her moral support and most importantly her friendship. We started out in Honours together as strangers and now she has become one of my closest friends. She has always stayed positive and encouraged me to keep going when I couldn’t and for the most part of it she has kept me sane and entertained. Thank you to Carol, Arlene, Mel, Sarah and Sindy, for your friendship, advice and support. I would especially like to thank my old and new friends who have been in awe of the work I do and have supported me most of all thank you for putting up with my crazy antics. In particular my friends who are like family Tia, Ash, Nadine, Daunia and Elise you have all kept me sane and in check and are always down for DnMs, lots of love. I would also like to thank my sister Jess and my brother Aaron thank you for all your love and support. We have gone through so much together as a family no matter what I will always love you both. Finally to Dad, I owe you so much, you have always supported my decisions in life and I’m grateful for everything you have given me. I love you so much! Table of Contents List of Tables............................................................................................................ vii List of Figures ............................................................................................................. x List of Abbreviations................................................................................................ xii Abstract ................................................................................................................... xvi Chapter 1: General Introduction...................................................................................... 1 1.1. Disease modelling using human pluripotent stem cells ................................... 2 1.1.1. Best vitelliform macular dystrophy ........................................................... 2 1.1.2. An unlimited source of human cells from human pluripotent stem cells . 3 1.1.3. Human pluripotent stem cells: a new paradigm for disease modelling .... 7 1.2. Importance of the RPE ..................................................................................... 8 1.2.1. RPE function ............................................................................................. 8 1.2.1.1. Absorption of light .............................................................................. 8 1.2.1.2. Transepithelial transport and ion homeostasis in the subretinal space 10 1.2.1.3. Phagocytosis of shed photoreceptor outer segments ......................... 11 1.3. Bestrophin-1 protein in focus ......................................................................... 13 1.3.1. The structure and function of the Bestrophin protein ............................. 13 1.3.2. Electrophysiological reports of BEST1 function .................................... 15 1.3.2.1. BEST1 a newly defined Cl- channel activated by Ca2+ ..................... 15 1.3.2.2. BEST1 plays an integral role in Ca2+ regulation ............................... 17 i 1.4. Hypothesis and Aims ...................................................................................... 19 1.4.1. Hypothesis ............................................................................................... 19 1.4.2. Aims ........................................................................................................ 19 Chapter 2: General Materials and Methods .................................................................. 20 2.1. Human ES cell lines ........................................................................................... 21 2.1.1. Cell line details ............................................................................................ 21 2.1.2. Genomic characterisation of the GENEA069 cells ..................................... 21 2.1.3. Human ES cell line maintenance ................................................................ 22 2.1.4. Human ES cell differentiation to RPE ........................................................ 23 2.1.5. Isolating human ES cell-derived RPE ......................................................... 24 2.2. Quantitative polymerase chain reaction ............................................................. 25 2.2.1. RNA collection............................................................................................ 25 2.2.2. cDNA synthesis via reverse transcription ................................................... 26 2.2.3. Primer design .............................................................................................. 27 2.2.4. PCR amplification of mRNA transcripts .................................................... 28 2.2.5. Real-time PCR ............................................................................................ 28 2.2.6. Analysis of relative quantitation of gene expression .................................. 29 2.3. Flow cytometry .................................................................................................. 30 2.3.1. Antibody staining for flow cytometry ......................................................... 30 2.3.2. Flow cytometry data acquisition ................................................................. 31 2.4. Colony forming assay for undifferentiated human ES cells .............................. 32 2.5. Teratoma Assay .................................................................................................. 32 ii 2.6. Immunofluorescence .......................................................................................... 33 2.7. Electrophysiology .............................................................................................. 34 2.7.1. Whole-cell patch clamp data collection ...................................................... 34 2.7.2. Solutions ...................................................................................................... 35 2.7.2.1. Whole-cell Na+ and K+ currents ............................................................... 35 2.7.2.2. Whole-cell Cl- and Ca2+ currents ............................................................. 36 2.7.3. Analysis of whole-cell patch clamp data .................................................... 37 2.8. Affymetrix gene expression analysis ................................................................. 38 2.8.1. Affymetrix microarray analysis .................................................................. 38 2.8.2. Identifying RPE gene expression data ........................................................ 38 2.8.3. Analysis of published human foetal and adult RPE datasets ...................... 39 2.8.4. Identifying Gene ontology categories in human ES cell-derived RPE ....... 39 2.8.5. Identifying ion channels in the expressed gene lists ................................... 40 Chapter 3: Characterisation of a novel human ES cell line containing the F305S BEST1 mutation ............................................................................................................ 41 3.1. Introduction .................................................................................................... 42 3.2. Results ............................................................................................................ 44 3.2.1. GENEA069 cells possess the disease-causing F305S BEST1 mutation 44 3.2.2. F305S-mutant BEST1 human ES cells display typical pluripotency phenotypes ............................................................................................................ 44 3.2.3. F305S-mutant BEST1 human ES cells are functional human ES cells .. 45 3.2.4. F305S-mutant BEST1 human ES cells form teratomas .......................... 46 iii 3.2.5. Characterisation of F305S-mutant BEST1 RPE relative to normal RPE50 3.2.6. Altered BEST1 localisation in the F305S-mutant BEST1 RPE.............. 51 3.2.7. F305S-mutant BEST1 and normal RPE have similar Affymetrix gene expression .............................................................................................................. 54 3.2.8. GO analysis reveals biological processes in human ES cell-derived