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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