MicroRNA Regulation of Chondrogenesis in Human Embryonic Stem Cells 2016 Rosie Sarah Griffiths A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Biology, Medicine and Health Supervisors: Prof Sue KIMBER and Dr Matthew RONSHAUGEN SCHOOL of BIOLOGICAL SCIENCES Division of Cell Matrix Biology & Regenerative Medicine Contents List of Figures 8 List of Tables 9 List of Abbreviations 11 Abstract 12 Declaration 13 Copyright Statement 14 Acknowledgements 15 Dedication 16 1 Introduction 17 1.1 General Overview . 17 1.2 Articular Cartilage Cell Therapy . 17 1.3 Chondrogenesis . 20 1.3.1 Signaling Pathways Regulating Chondrogenesis . 21 1.3.2 Transcriptional Regulation of Chondrogenesis . 22 1.4 Embryonic Stem Cells . 23 1.4.1 Pluripotency control . 23 1.4.2 Extrinsic Factors Promoting Self-Renewal in ESCs . 24 1.4.3 ESC Characterisation . 25 1.4.4 ESC Culture . 25 1.4.5 Feeder-Free Culture . 26 1.5 MicroRNAs . 26 1.5.1 MicroRNAs in the Genome . 27 1.5.2 Biogenesis . 27 1.5.3 MicroRNA Silencing Mechanism . 30 1.5.4 Target Identification . 30 2 MicroRNA Regulation of Chondrogenesis in Human Embryonic Stem Cells 1.5.5 MicroRNAs in ESCs . 32 1.5.6 MicroRNAs involved in Differentiation . 33 1.5.7 Chondrogenic miRNAs . 34 1.5.8 Manipulating miRNAs . 39 1.6 Next Generation Sequencing and Bioinformatic Analysis . 41 1.6.1 Mapping to miRNAs . 42 1.6.2 Normalisation . 42 1.6.3 Differential Expression of miRNAs . 42 1.6.4 Co-expression Analysis . 43 1.7 Exosomes . 44 1.7.1 Exosome Isolation . 45 1.7.2 Exosome Characterisation and Quantification . 46 1.7.3 Exosome Composition . 47 1.7.4 Exosome Biogenesis . 48 1.7.5 Exosome Release . 51 1.7.6 Exosome Uptake . 52 1.7.7 Exosome Function . 52 1.7.8 Exosomal miRNAs . 54 1.7.9 Clinical Applications of Exosomes . 55 1.8 Research Aims . 57 2 Materials and Methods 58 2.1 Mouse Embryonic Fibroblast (MEF) Culture . 58 2.1.1 Defrosting active MEFs . 58 2.1.2 Culturing active MEFs . 58 2.1.3 Passaging active MEFs with TrypLE . 58 2.1.4 MEF Inactivation . 59 2.1.5 Inactivated MEF plating . 59 2.2 Human Embryonic Stem Cell culture . 60 2.2.1 Human Embryonic Stem Cell derivation . 60 2.2.2 Defrosting and Feeding hESCs . 60 2.2.3 Passaging hESCs with TrypLE . 61 2.2.4 Passaging hESCs with EDTA . 61 2.2.5 Feeder Free Culture of hESCs . 62 2.2.6 Chondrogenic differentiation of hESCs . 62 2.3 RNA Analysis . 63 2.3.1 RNA Extraction . 63 2.3.2 MicroRNA TaqMan Real-Time Polymerase Chain Reaction (RT-PCR) 63 Chapter 0 3 MicroRNA Regulation of Chondrogenesis in Human Embryonic Stem Cells 2.3.3 SYBR Green RT-PCR . 64 2.4 RNA Sequencing analysis . 64 2.4.1 RNA-Seq library preparation . 64 2.4.2 Galaxy . 65 2.4.3 Mapping miRNAs to miRBase . 65 2.4.4 Finding differentially expressed miRNAs . 66 2.4.5 miRComb . 66 2.5 Exosome methods . 67 2.5.1 Exosome free media preparation . 67 2.5.2 Exosome Isolation . 67 2.5.3 Exosomal RNA isolation . 68 2.5.4 Exosomal RNA quantification . 68 2.5.5 Dynamic Light Scattering . 69 2.5.6 Exosome labelling with PKH26 dye . 69 2.5.7 Electron microscopy of Exosomes . 69 2.5.8 Cartilage Digestion and Cartilage Exosome Isolation . 70 2.6 Molecular biology . 71 2.6.1 Agarose gel electrophoresis . 71 2.6.2 Ligations . 71 2.6.3 Transformation of plasmids into competent E.coli . 71 2.6.4 Plasmid Verification . 71 2.6.5 CD63-eGFP Fusion Protein plasmid . 72 2.7 Third Generation Lentiviral Production . 73 2.7.1 HEK293T cell culture . 73 2.7.2 Plasmid transduction . 73 2.7.3 Lentivirus collection and isolation . 74 2.7.4 Lentivirus quantification . 74 3 Results I - Whole Transcriptome and Small RNA-seq analysis of Chondrogenesis in hESCs 76 3.1 Aims and Introduction . 76 3.2 Results . 77 3.2.1 Small RNA-seq quality control . 77 3.2.2 Changes in miRome and Transcriptome variation during hESC directed chondrogenesis . 79 3.2.3 Highest expressed miRNAs in hESCs and hESC-derived chondroprogenitor cells . 83 Chapter 0 4 MicroRNA Regulation of Chondrogenesis in Human Embryonic Stem Cells 3.2.4 Differential expression analysis of miRome and transcriptome of hESCs undergoing directed chondrogenesis . 84 3.2.5 Biological variability in hESC directed chondrogenesis can be exploited to identify novel miRNAs . 89 3.2.6 Stage-wise differential expression analysis miRNAs . 92 3.3 Discussion . 97 3.3.1 Small RNA-seq technical variation . 97 3.3.2 Cell line variation . 98 3.3.3 Highest expressed miRNAs during hESC directed chondrogenesis . 99 3.3.4 Summary . 101 4 Results II - Integrated miRomics and Transcriptomics analysis 103 4.1 Introduction and Aims . 103 4.2 Results . 104 4.2.1 Correlation Analysis . 104 4.2.2 Protein Network Analysis . 109 4.2.3 MicroRNA Target Interaction Network . 113 4.2.4 MicroRNA Functional Studies- miR-199a Inhibition . 116 4.3 Discussion . 117 4.3.1 Identification of novel regulators during hESC-directed chondrogenesis by co-expression network analysis . 117 4.3.2 MicroRNA Target Interaction Analysis . 127 4.3.3 Conclusion . 127 5 Results III - Exosomes 129 5.1 Aims and Introduction . 129 5.2 Results . 129 5.2.1 Exosome validation . 129 5.2.2 Expression of miR-302a in Pluripotent stem cell derived exosomes . 131 5.2.3 Exosome qPCR optimisation . 132 5.2.4 Exosomal miRNAs in hESC directed chondrogenesis . 138 5.2.5 Exosomal Sequencing Quality . 139 5.2.6 RNA-seq Cluster Analysis . 142 5.2.7 Exosome-enriched miRNAs from hESCs and chondroprogenitors . 144 5.2.8 Exosomal miRNA motif enrichment . 146 5.2.9 Pathway analysis of exosomal enriched miRNAs . 146 5.2.10 Cartilage Exosomes . 149 5.2.11 Exosome Uptake and Localisation . 150 Chapter 0 5 MicroRNA Regulation of Chondrogenesis in Human Embryonic Stem Cells 5.2.12 High-throughput Exosome Uptake.