Investigating the effect of plant-derived extracellular vesicles on human placental function A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2018 Kate M Timms School of Medical Sciences 1 Contents Contents .................................................................................................................................... 2 List of tables .............................................................................................................................. 8 List of Figures ............................................................................................................................ 9 Abbreviations .......................................................................................................................... 12 Abstract of thesis. ................................................................................................................... 14 Declaration .............................................................................................................................. 15 Copyright statement ............................................................................................................... 15 Dedication ............................................................................................................................... 16 Acknowledgements ................................................................................................................. 16 Introduction ................................................................................................ 17 1.1 Introduction ................................................................................................................ 18 1.2 Physiological and pathological development and function of the placenta ............... 19 1.2.1 Development and function of trophoblast in early pregnancy .......................... 19 1.2.2 The differentiation and role of trophoblast lineages in placental development and function ........................................................................................................................ 21 1.2.2.1 Placental nutrient transport ........................................................................... 25 1.3 Role of maternal nutrition in placental and fetal growth ........................................... 26 1.3.1 Fruit and Vegetable extracellular vesicles as potential nutrients ....................... 29 1.3.1.1 Evidence for the intestinal uptake of dietary extracellular vesicles ............... 32 1.3.1.2 Dietary uptake of plant extracellular vesicle components and their potential roles in human health ..................................................................................................... 36 1.3.1.2.1 Dietary derived miRNAs ............................................................................ 37 1.4 Intestinal-placental communication ........................................................................... 39 1.5 Hypothesis ................................................................................................................... 40 1.6 Aims............................................................................................................................. 40 Materials and Methods ............................................................................... 42 2.1 Materials ..................................................................................................................... 43 2.2 Watermelon selection ................................................................................................. 43 2.3 Cell culture .................................................................................................................. 43 2.3.1 Caco-2 differentiation ......................................................................................... 45 2.3.2 Lucifer yellow rejection as a measure of Caco-2 barrier tightness ..................... 45 2.4 Isolation of exosomes and extracellular vesicles ........................................................ 45 2.4.1 Isolation of extracellular vesicles from watermelon ........................................... 45 2.4.1.1 Watermelon pre-processing ........................................................................... 45 2.4.1.2 Watermelon extracellular vesicle isolation ..................................................... 46 2.4.2 Isolation of exosomes from human cell-lines ..................................................... 46 2 2.5 Identification of extracellular vesicle size, morphology and concentration ............... 47 2.5.1 Extracellular vesicle sizing and quantification .................................................... 47 2.5.2 Analysis of EV morphology .................................................................................. 47 2.6 Fluorescent labelling of watermelon extracellular vesicles and Ishikawa exosomes . 48 2.6.1 PKH26-labelling ................................................................................................... 48 2.6.2 SYTO RNAselect-labelling .................................................................................... 50 2.7 Assessing uptake of fluorescently labelled extracellular vesicles and exosomes ....... 50 2.7.1 Extracellular vesicle and exosome uptake into human cells .............................. 50 2.7.1.1 Inhibition of watermelon extracellular vesicle and Caco-2 exosome uptake . 50 2.7.2 Watermelon extracellular vesicle uptake into small intestinal tissue obtained from pregnant mice ............................................................................................................ 52 2.7.2.1 Mouse housing, husbandry and small intestine collection ............................. 52 2.7.2.2 Incubation of mouse small intestines with watermelon extracellular vesicles 52 2.8 Immunocytochemistry ................................................................................................ 53 2.8.1.1 Use of ImageJ for automated detection of apoptotic nuclei .......................... 55 2.9 Flow cytometry ........................................................................................................... 57 2.10 Analysis of SYTO RNAselect labelled watermelon extracellular vesicle RNA passage from apical to basal media compartments ............................................................................. 59 2.11 Analysis of protein content by gel electrophoresis and Western blot ....................... 59 2.11.1 Cell and extracellular vesicle lysis ....................................................................... 59 2.11.2 Gel electrophoresis ............................................................................................. 60 2.11.3 Western blot ....................................................................................................... 60 2.12 miRNA profiling ........................................................................................................... 61 2.12.1 RNA Extraction .................................................................................................... 61 2.12.1.1 Watermelon cells and extracellular vesicles ............................................... 61 2.12.1.2 Caco-2 and BeWo cells ................................................................................ 62 2.12.2 Ligation and reverse transcription ...................................................................... 62 2.12.3 Optimisation of miScript qPCR for amplification of plant miRNA from extracellular vesicles ........................................................................................................... 63 2.12.4 Quality control of samples prior to array analysis .............................................. 64 2.12.5 Rice miFinder qPCR arrays .................................................................................. 65 2.12.5.1 Watermelon cells and extracellular vesicles ............................................... 65 2.12.5.2 Caco-2 and BeWo cells ................................................................................ 67 2.13 Lipid profiling of watermelon extracellular vesicles ................................................... 70 2.13.1 Sample preparation ............................................................................................ 70 2.13.2 UHPSFC/ESI-MS conditions ................................................................................. 72 Lipid identification and nomenclature ................................................................................ 72 3 2.14 Proteomics .................................................................................................................. 73 2.14.1 Samples ............................................................................................................... 73 2.14.1.1 Watermelon cells and extracellular vesicles ............................................... 73 2.14.1.2 Caco-2 basal medium .................................................................................. 73 2.14.1.3 Caco-2 exosomes ........................................................................................ 73 2.14.1.4 BeWo cells ................................................................................................... 73 2.14.2 Sample and gel top preparation ......................................................................... 74 2.14.3 Mass spectrometry for protein analysis ............................................................