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This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. MICRORNA REGULATION OF DRUG METABOLISM IN STEM CELL – DERIVED HEPATOCYTES DAGMARA SZKOLNICKA MSc University of Edinburgh 2015 College of Medicine and Veterinary Medicine The dissertation is submitted for the degree of Doctor of Philosophy 0 TABLE OF CONTENTS Abstract ..................................................................................................................................... 5 Declaration ................................................................................................................................ 7 List of Figures and Tables ......................................................................................................... 8 Abbreviations .......................................................................................................................... 12 Acknowledgments ................................................................................................................... 18 Publications ............................................................................................................................. 19 CHAPTER ONE: INTRODUCTION ....................................................................................... 20 1. Introduction ................................................................................................................... 21 1.1 Liver as an organ .................................................................................................... 21 1.2 Overview of the liver development ....................................................................... 24 1.2.1 Endoderm formation .......................................................................................... 25 1.2.2 Foregut specification .......................................................................................... 27 1.2.3 Hepatic specification .......................................................................................... 28 1.2.4 Liver bud formation ........................................................................................... 28 1.2.5 Hepatocyte differentiation and maturation ......................................................... 29 1.2.6 Fetal, neonatal, and adult liver ............................................................................ 30 1.3 Liver diseases .......................................................................................................... 33 1.4 Modelling liver biology and disease in vitro ......................................................... 34 1.5 Pluripotent stem cells (PSCs) as renewable somatic cell-based models ............ 35 1.5.1 Using defined cell niches to stabilize cell phenotype ....................................... 37 1.5.2 Recent advances in generation of hepatocytes .................................................. 38 1.5.3 ‘Off the shelf’ cell – based therapies.................................................................. 39 1.6 Drug metabolism .................................................................................................... 40 1.6.1 Phase I ............................................................................................................... 43 1.6.2 Phase II .............................................................................................................. 46 1.6.3 Phase III ............................................................................................................ 54 1.6.4 Drug – induced liver injury and current biomarkers ......................................... 58 1 1.7 MicroRNAs ...................................................................................................................... 59 1.7.1 MicroRNA biogenesis and function .................................................................. 61 1.7.2 The role of microRNAs in liver diseases and drug-induced liver injury .......... 63 1.7.3 Computer programmes to predict microRNA binding ....................................... 64 1.8 The objectives of the thesis .................................................................................. 65 CHAPTER TWO: MATERIALS AND METHODS ................................................................ 68 2.1 Materials and Solutions ....................................................................................... 69 2.1.1 Reagent solutions/chemicals/SDS gels ............................................................. 69 2.1.2 Pluripotent stem cells ........................................................................................ 69 2.1.3 Cell culture media, factors, reagents and culture plates .................................... 70 2.1.4 Media and supplement preparation for each cell line maintenance and differentiation stage .................................................................................... 71 2.1.5 Antibodies ......................................................................................................... 72 2.1.6 Primers .............................................................................................................. 74 2.1.7 MicroRNA precursors and antagomirs ............................................................. 75 2.2 Mammalian cell culture and differentiation ...................................................... 76 2.2.1 Maintenance of pluripotent stem cells .............................................................. 76 2.2.2 Embryoid body (EB) formation ........................................................................ 80 2.2.3 Hepatic differentiation of pluripotent stem cells ............................................... 80 2.3 Characterisation of pluripotent stem cells (PSCs) and PSCs-derived hepatocytes ............................................................................ 83 2.3.1 Immunocytochemistry ....................................................................................... 83 2.3.2 ELISA Assays ................................................................................................... 83 2.3.3 Cytochrome P450 Assays ................................................................................. 84 2.3.4 Analysis of single nucleotide polymorphism (SNP) in pluripotent stem cells .. 85 2.3.5 Fluorescence activated cell sorting (FACS) ...................................................... 86 2.4 Molecular techniques ........................................................................................... 87 2.4.1 RNA isolation and extraction ............................................................................ 87 2.4.2 Reverse transcription and polymerase chain reaction (PCR) ............................ 88 2.4.3 Reverse transcription and RT2 profiler PCR array (Human Drug Metabolism) 89 2.4.4 MicroRNA microarray ...................................................................................... 91 2 2.5 Protein biochemistry techniques .......................................................................... 93 2.5.1 Cellular protein extraction ................................................................................. 93 2.5.2 Measuring protein concentration ....................................................................... 93 2.5.3 SDS – NuPage® polyacrylamide gel electrophoresis ........................................ 93 2.5.4 Western immunoblotting ................................................................................... 94 2.6 Modelling paracetamol toxicity in vitro .............................................................. 95 2.7 RNAi transfection techniques ............................................................................. 96 2.8 Modulating paracetamol toxicity ........................................................................ 98 2.9 Plasma sample collection, processing, patient information .............................. 99 2.10 Fulminant Plasma Experiments ...................................................................... 100 2.11 Compound Incubation (DILI Assays) ............................................................ 100 2.12 Statistical analysis ............................................................................................ 101 2.12.1 Student’s t-test ............................................................................................... 101 2.12.2 Multi-group comparison of different cell types ............................................ 101 2.12.3 RT2 profiler PCR array data analysis ............................................................ 102 2.13 miR-mRNA binding analysis .......................................................................... 102 CHAPTER THREE: PLURIPOTENT STEM CELL MAINTENANCE AND HEPATOCELLULAR DIFFERENTIATION
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