Simplified In Vitro Engineering of Functional Mammalian Neuromuscular Junctions between Embryonic Rat Motor Neurons and Immortalised Human Skeletal Muscle Cells JASDEEP SAINI A thesis submitted in partial fulfilment of the requirements of Manchester Metropolitan University for the degree of Doctor of Philosophy Department of Life Sciences Manchester Metropolitan University 2019 Table of Contents i Acknowledgements iv Publications v List of Abbreviation vi List of Figures x List of Tables xiii Abstract xiv Chapter 1: Introduction 1.0 Myogenesis 1 1.0.0 Embryonic Myogenesis 1 1.0.1 Adult Myogenesis 4 1.1 Skeletal Muscle 7 1.1.0 Anatomy 7 1.1.1 Skeletal Muscle Fibres 9 1.1.2 Skeletal Muscle Fibre Types 11 1.1.3 Muscle Contraction 12 1.2 Motor Neurons 17 1.2.0 Motor Neuron Subtype Diversity 17 1.3 The Neuromuscular Junction 22 1.3.0 Agrin 22 1.3.1 Muscle-Specific Tyrosine Kinase 23 1.3.2 43 kDa Receptor-Associated Protein of the Synapse 26 1.3.3 Acetylcholine Receptors 26 1.3.4 Neuromuscular Junction Function 27 1.4 Aims and Objectives 32 Chapter 2: Materials and Methods 2.0 Materials 33 2.0.0 Cell Lines 33 2.0.1 Animals 33 2.0.2 Laboratory Equipment 34 2.0.3 Laboratory Plasticware 34 2.0.5 Reagents 35 2.1 Methods 36 2.1.0 Human Skeletal Muscle Cell Culture 36 2.1.1 Cell Count 37 2.1.2 Subculture 38 2.1.3 Cryopreservation 38 2.1.4 Differentiation Parameters 39 2.1.5 Co-culture 41 2.1.6 Isolation of Rat Embryo Spinal Cord 43 2.1.7 Preparation of Skeletal Muscle Cells for Co-culture 43 2.1.8 Innervation of Skeletal Muscle with Embryonic Rat Motor Neurons 43 2.1.9 Differentiation Parameters: Co-culture 44 2.1.10 Disassociation of Rat Embryo Spinal Cord 44 2.1.11 Rat agrin ELISA 45 2.1.12 Co-culture Fixation 46 2.1.13 Co-culture Immunocytochemistry 46 i 2.1.14 Quantification of Neuromuscular Junction Morphologies 48 2.1.15 Quantification of Transversal Triad Formation 48 2.1.16 Quantification of Peripheral Myonuclei 49 2.1.17 Quantification of Striation Formation 49 2.1.18 Quantification of Myotube Thickness 49 2.1.19 NMJ Functionality Assessed via Measuring Spontaneous Contractions 50 2.1.20 Human Growth Factor Array 51 2.1.21 Statistical Analysis 53 Chapter 3: Proliferation and Differentiation of Young and Old Immortalised Human Myoblasts. 3.0 Background 54 3.0.0 Introduction 54 3.0.1 Aims 55 3.1 Results 56 3.1.0 Myoblast Revival 56 3.1.1 Myoblast Proliferation 58 3.1.2 Myoblast Viability 60 3.1.3 Optimisation of Myoblast Seeding Density 62 3.1.4 Optimisation of Differentiation Media 64 3.1.5 Differentiation Parameters 67 3.1.6 Proliferation & Differentiation Marker Expression 69 3.2 Discussion 72 3.3 Conclusions 75 Chapter 4: A Novel System of Immortalised Human Myoblasts Co-cultured with Rat Embryonic Spinal Cord Explants 4.0 Background 76 4.0.0 Introduction 76 4.0.1 Aims 78 4.1 Results 79 4.1.0 Isolation of Embryonic Rat Spinal Cord 79 4.1.1 Viability of Spinal Cord Explants 83 4.1.2 Validation of Neuron Proliferation 85 4.1.3 Characterisation of Co-culture Morphology 87 4.1.4 Spontaneous Myotube Contractions 91 4.1.5 Spinal Cord Explant Co-culture vs Disassociated Spinal Cord Co-culture 92 4.1.6 Expression of Rat Agrin 94 4.1.7 Preliminary Neuromuscular Junction Formation 96 4.2 Discussion 98 4.3 Conclusions 100 Chapter 5: Characterisation of in vitro Neuromuscular Junctions between Embryonic Rat Motor Neurons and Immortalised Human Myoblasts 5.0 Background 101 5.0.0 Introduction 101 5.0.1 Aims 103 5.1 Results 104 5.1.0 Optimisation of Co-cultures for Characterisation 104 ii 5.1.1 Characterisation of Cholinergic Motor Neurons 106 5.1.2 Characterisation of Neuroglia 109 5.1.3 Characterisation of NMJ formation 112 5.1.4 Characterisation of Presynaptic Activity 116 5.1.5 Characterisation of Postsynaptic Elements 118 5.1.6 Characterisation of Innervated Myotubes 120 5.2 Discussion 126 5.3 Conclusion 128 Chapter 6: Functional Assessment of in vitro Neuromuscular Junctions between Embryonic Rat Motor Neurons and Immortalised Human Myoblasts 6.0 Background 129 6.0.0 Introduction 129 6.0.1 Aims 131 6.1 Results 132 6.1.0 Functional Assessment of NMJs with α-Bungarotoxin 132 6.1.1 Functional Assessment of NMJs with Tubocurarine 135 6.1.2 Functional Assessment of NMJs with Bicuculline 137 6.1.3 Functional Assessment of NMJs with L-Glutamic Acid 139 6.1.4 Functional Assessment of NMJs with γ-Aminobutyric Acid 141 6.2 Discussion 143 6.3 Conclusion 145 Chapter 7: Investigation of Growth and Neurotrophic Factor Concentrations in Co-Cultures of Human Myoblasts Innervated by Rat Embryonic Spinal Cord Explants Compared with Human Myoblast Monocultures. 7.0 Background 146 7.0.0 Introduction 146 7.0.1 Aims 148 7.1 Results 149 7.1.0 Quantification of Growth and Neurotrophic Factors 149 7.2 Discussion 151 7.3 Conclusion 154 Chapter 8: General Discussion & Conclusions 8.0 Discussion 155 8.1 Conclusion & Future Directions 162 Chapter 9: References 9.0 References 163 Appendix Appendix A 210 Appendix B 211 iii Acknowledgements Firstly, thank you to my director of studies Dr Nasser Al-Shanti for the opportunity to pursue my academic career and all the support and guidance throughout this project. Special thanks to Dr Jamie S McPhee, Prof Hans Degens, Dr Alessandro Faroni, and Dr Adam J Reid for their help and time throughout this project. Most importantly, thank you to my father (Harnek Singh), mother (Daljit Kaur), and wife (Seema Saini) for their past, present, and future support. Lastly, thank you to my son Aary Saini for motivating me to pursue excellence in all aspects of my life. iv Publications Original Research Paper Saini, J., Faroni, A., Abd Al Samid, M., Reid, A. J., Lightfoot, A. P., Mamchaoui, K., Mouly, V., Butler- Browne, G., McPhee, J. S., Degens, H. and Al-Shanti, N. (2019) 'Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells.' Stem Cells Cloning, 12 pp. 1-9. Review Saini, J., McPhee, J. S., Al-Dabbagh, S., Stewart, C. E. and Al-Shanti, N. (2016) 'Regenerative function of immune system: Modulation of muscle stem cells.' Ageing Res Rev, 27, May, pp. 67-76. Contributing Author Abd Al Samid, M., McPhee, J. S., Saini, J., McKay, T. R., Fitzpatrick, L. M., Mamchaoui, K., Bigot, A., Mouly, V., Butler-Browne, G. and Al-Shanti, N. (2018) 'A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts.' Stem Cells Cloning, 11 pp. 85-93. v List of abbreviations ± SD plus/minus standard deviation α-BTX α-bungarotoxin αFF fast-twitch fatigable motor neurons αFR fast-twitch fatigue-resistant motor neurons αMN(s) alpha motor neuron(s) αS slow-twitch fatigue resistant motor neurons β-III-tubulin class III β-tubulin βMN(s) beta motor neuron(s) γMN(s) gamma motor neuron(s) AP(s) action potential(s) ACh acetylcholine AChR(s) acetylcholine receptor(s) ADP adenosine diphosphate ALS amyotrophic lateral sclerosis AR aspect ratio ATM 37°C with a 5% CO2 atmosphere ATP adenosine triphosphate BDNF brain-derived neurotrophic factor bHLH basic helix–loop–helix C25 25-year-old immortalised human myoblasts C83 83-year-old immortalised human myoblasts Ca2+ calcium ions CF contraction frequency ChAT choline acetyltransferase CMS congenital myasthenic syndrome CNS central nervous system DAPI 4′,6-Diamidine-2′-phenylindole dihydrochloride DHPR dihydropyridine receptor DM differentiation media DMEM Dulbecco’s modified eagle medium DMSO Dimethyl sulfoxide vi Dok7 docking protein 7 DPBS Dulbecco’s phosphate buffered saline 1X DRG(s) dorsal root ganglion(s) DS donkey serum DSC disassociated spinal cord ECM extracellular matrix ED embryonic development day EGF(s) epidermal growth factor(s) EPP endplate potential FBS fetal bovine serum FGF-7 fibroblast growth factor 7 FGF(s) fibroblast growth factor(s) FGFb basic fibroblast growth factor FI fusion index GABA γ-aminobutyric acid GDF-11 growth differentiation factor 11 GDNF glial-cell-line-derived neurotrophic factor GFAP glial fibrillary acidic protein GM complete growth media GS goat serum HBSS Hanks' balanced salt solution hESC(s) human embryonic stem cell(s) HGF(s) Hepatocyte growth factor(s) hiPSC(s) human induced pluripotent stem cell(s) HMC(s) hypaxial motor column(s) HS horse serum ICC immunocytochemistry IGF-1 insulin-like growth factor 1 IGFBP(s) insulin-like growth factor-binding protein(s) IP inorganic phosphate K+ potassium ions LEMS Lambert-Eaton myasthenic syndrome L-Glut L-glutamic acid LMC(s) lateral motor column(s) vii LMCl lateral lateral motor column LMCm medial lateral motor column LRP4 low-density lipoprotein receptor-related protein 4 MA myotube area MEP(s) motor endplate(s) MMC(s) medial motor column(s) MN(s) motor neuron(s) MNT(s) motor neuron terminal(s) MHC myosin heavy chain MRF4 myogenic regulatory factor 4 MRF(s) myogenic regulatory factor(s) MG myasthenia gravis MuSK muscle-specific tyrosine kinase Myf5 myogenic factor 5 Na+ sodium ions ND neurodegenerative NFH neurofilament heavy NGF nerve growth factor NM neuromuscular NMJ(s) neuromuscular junction(s) NT3 neurotrophin 3 NT4 neurotrophin 4 NT5 neurotrophin 5 Pax3 paired box protein 3 Pax7 paired box protein 7 Pen/Strep penicillin-streptomycin PGC preganglionic column PlGF placental growth factor PMC phrenic motor column PWB Prem/Wash buffer Rapsyn 43 kDa receptor-associated protein of the synapse RyR ryanodine receptor SC(s) satellite cell(s) SCE(s) spinal cord explants viii SkM skeletal muscle SkMC(s) skeletal muscle cell(s) SMA spinal muscular atrophy SMN1 survival motor neuron 1 SMN2 survival motor neuron 2 SOD1 superoxide dismutase 1 SR sarcoplasmic reticulum Sty1 synaptotagmin 1 T-tubule transverse tubule TGFβ transforming growth factor beta TX100 Triton X-100 VAChT vesicular acetylcholine transporter VEGF vascular endothelial growth factor ix List of Figures Figure 1.0: Schematic representation of somite maturation underlining 3 embryonic myogenesis Figure 1.1: Adult myogenesis.