Skeletal Muscle Stem Cells: Methods and Protocols

Preface
Contents
Contributors
Part I: Muscle Stem & Progenitor Cells
Chapter 1: Flow Cytometer Analyses, Isolation, and Staining of Murine Muscle Satellite Cells
1 Introduction
2 Materials
2.1 Preparation of Mononuclear Cells from Murine Skeletal Muscles
2.2 Staining of Mononuclear Cells and Isolation of Muscle Satellite Cells
2.3 Staining of Isolated Muscle Satellite Cells
3 Methods
3.1 Preparation of Mononuclear Cells from Murine Skeletal Muscles
3.2 Staining of Mononuclear Cells and Isolation of Satellite Cells
3.3 FACS Analyses
3.4 Staining of Isolated MuSCs (See Fig. 3)
4 Notes
References
Chapter 2: Extra Eyelid-Derived Muscle Stem Cells
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Cells for Primary Culture
3.2 Preparation of Cultured Myogenic Cells for Cell Sorting
3.3 Characterization of Isolated Cells Ex Vivo
3.4 Immunocytochemical Analysis
4 Notes
References
Chapter 3: Isolation, Culture, and Analysis of Zebrafish Myofibers and Associated Muscle Stem Cells to Explore Adult Skeletal ...
1 Introduction
2 Materials
2.1 Materials for Dissection and Dissociation of Adult Zebrafish Muscle
2.2 Materials Required for Myofiber Isolation and MuSC Culture
2.3 Materials Required for Myofiber and MuSC-Derived Cells Immunostaining
3 Method
3.1 Preparation
3.2 Muscle Dissection
3.3 Myofiber Dissociation and Isolation
3.4 Morphometric Analysis of Isolated Myofibers
3.5 Immunostaining of Isolated Myofibers
3.6 Myofiber-Derived MuSC Culture and Immunostaining
4 Notes
References
Chapter 4: The Satellite Cell Colony Forming Cell Assay as a Tool to Measure Self-Renewal and Differentiation Potential
1 Introduction
2 Materials
2.1 Harvest of Satellite Cells for FACS Sorting
2.2 FACS Staining
2.3 Antibody Staining of Colonies
3 Methods
3.1 Preparing the Cell Culture Plates
3.2 Harvest of Satellite Cells
3.3 Antibody Staining (Optional)
3.4 FACS Gating Strategy and Single Cell Sorting
3.5 Cell Culture, Staining, and Imaging
4 Notes
References
Chapter 5: Co-cultures of Macrophages with Muscle Stem Cells with Fibroadipogenic Precursor Cells from Regenerating Skeletal M...
1 Introduction
2 Materials
2.1 Cell Sorting and Culture
2.2 Immunostaining
3 Methods
3.1 Digestion
3.1.1 Single Digestion
3.1.2 Double Digestion (from)
3.2 MACS Cell Sorting
3.2.1 MPs: CD64pos MACS Cell Sorting (see Note 6)
3.2.2 MuSCs: MACS Negative Sorting
3.2.3 FAPs: Isolation via Preplating
3.3 FACS Isolation (Fig. 1)
3.4 Co-cultures (Fig. 1)
3.4.1 Direct Co-cultures
Co-culture of MPs with MuSCs
Co-culture of MPs with FAPs
3.4.2 Indirect Co-culture (with inserts)
Co-culture of FAPs with MuSCs
Co-culture of FAPs with MPs
3.5 Immunostainings
4 Notes
References
Chapter 6: Measuring Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Muscle Stem Cells Using a Se...
1 Introduction
2 Materials
2.1 Isolation of Satellite Cells by Fluorescence-Activated Cell Sorting (FACS)
2.2 Seahorse Plate Preparation
2.3 Seahorse Experiment Preparation
2.4 Seahorse Analysis
2.5 Normalization
2.6 Data Analysis
3 Methods
3.1 Satellite Cells´ Isolation by FACS
3.2 Seahorse Plate Preparation and Seeding
3.3 Seahorse Experiment Preparation
3.4 Seahorse Analysis
3.5 Normalization
3.6 Data Analysis
4 Notes
References
Chapter 7: High Throughput Screening of Mitochondrial Bioenergetics in Myoblasts and Differentiated Myotubes
1 Introduction
2 Materials
2.1 Isolation and Culture of Primary Satellite Cells (Myotubes) and C2C12 Myoblasts
2.2 Extracellular Flux Assay
3 Methods
3.1 Isolation of Satellite Cells Using the Single Fiber Method
3.2 Preparation of Extracellular Flux Assay (the Day Before Assay)
3.3 Preparation of Extracellular Flux Assay (Day of the Assay)
3.4 Preparation of Compounds, Loading of Compounds to Each Port, and Analysis
4 Notes
References
Chapter 8: State of the Art Procedures for the Isolation and Characterization of Mesoangioblasts
1 Introduction
2 Materials
2.1 Basic Materials
2.2 Media
2.3 Antibodies (See Table 1)
3 Methods
3.1 Adult MAB Isolation
3.2 Human Fetal MAB Isolation
3.3 Murine Adult MAB Isolation by Fluorescence-Activated Cell Sorter (FACS)
3.4 Human Adult MAB (hMAB) Isolation by FACS
3.5 Human Fetal MAB (hfMAB) Isolation by FACS
3.6 MAB Batches Long-Term Storage
3.7 Murine Adult MAB Isolation by FACS for Single Cell RNA Sequencing (scRNA-seq)
3.8 scRNA-seq Analysis
3.9 Collagen Coating
3.10 Cell Fusion Potential: MABs and C2C12 Cell Cocultures
3.11 MAB Smooth Muscle Induction
3.12 MAB Osteogenic Induction
3.13 MAB Adipogenic Induction
3.14 MAB Chondrogenic Induction
4 Notes
References
Chapter 9: Analyses of Mesenchymal Progenitors in Skeletal Muscle by Fluorescence-Activated Cell Sorting and Tissue Clearing
1 Introduction
2 Materials
2.1 Dissociating Cells from Skeletal Muscle
2.2 Purification of Mesenchymal Progenitors and Satellite Cells by FACS
2.3 Whole-Mount Immunofluorescent Imaging of Mesenchymal Progenitors
3 Methods
3.1 Dissociating Cells from Skeletal Muscle
3.2 Purification of Mesenchymal Progenitors and Satellite Cells by FACS
3.2.1 Antibody Staining for FACS
3.2.2 Cell Sorting of Mesenchymal Progenitors and Satellite Cells
3.3 Whole-Mount Immunofluorescent Imaging of Mesenchymal Progenitors
3.3.1 Making Silicone Rubber Plate for Tissue Fixation
3.3.2 Whole-Mount Immunofluorescent Staining of Mouse Skeletal Muscle
4 Notes
References
Chapter 10: In Vitro Maturation of Human Pluripotent Stem Cell-Derived Myotubes
1 Introduction
2 Materials
2.1 Pluripotent Stem Cell Culture
2.2 Lentivirus Production and Transduction of PS Cells
2.3 Myogenic Differentiation of PS Cells
2.4 Differentiation of Myogenic Progenitors into Myotubes
3 Methods
3.1 Generation of iPAX7-hPS Cells
3.2 Myogenic Differentiation of iPAX7-hPS Cells
3.3 Optimized Protocol for Enhanced Myotube Differentiation/Maturation
4 Notes
References
Chapter 11: Differentiation of Human Fetal Muscle Stem Cells from Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 HiPSC Lines and Maintenance of Feeder-Free Culture
2.2 Differentiation of Fetal Skeletal Muscle Stem Cells
2.3 Cell Preparation for FACS Analysis and Sorting
2.4 Immunocytochemistry
3 Methods
3.1 Feeder-Free Culture for hiPSCs
3.2 Differentiation of hiPSCs into Fetal MuSCs
3.2.1 Day -3: Cell Plating
3.2.2 Day 0: Initiate Mesodermal Differentiation
3.2.3 Day 7: Passage for the Dermomyotome-Like Cells Differentiation
3.2.4 Day 14: Passage for Myogenic Differentiation
3.2.5 Day 17: Promote Myogenic Differentiation
3.2.6 Day 38: Initiate Muscle Maturation
3.3 Cell Preparation for FACS Analysis and Sorting
3.4 Immunocytochemistry of the Sorted Cells
4 Notes
References
Chapter 12: Sphere-Based Expansion of Myogenic Progenitors from Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 EZ Sphere Preparation and Passaging
2.2 EZ Sphere Dissociation and Coverslip Plating for Terminal Differentiation
2.3 Immunocytochemistry
3 Methods
3.1 EZ Sphere Preparation from Human PSC Colonies
3.2 Passaging EZ Spheres by Mechanical Chopping
3.3 EZ Sphere Dissociation and Terminal Differentiation of Myogenic Progenitors
3.4 Immunocytochemistry to Confirm Myocytes and Myotubes
4 Notes
References
Chapter 13: Producing Engraftable Skeletal Myogenic Progenitors from Pluripotent Stem Cells via Teratoma Formation
1 Introduction
2 Materials
2.1 Cell Culture and Maintenance
2.2 Irradiation, Injury, and Transplantation
2.3 Cell Isolation
2.4 Fluorescence-Activated Cell Sorting (FACS)
2.5 Sectioning and Immunohistochemistry
3 Methods
3.1 Mouse Embryonic Stem Cells Preparation
3.2 Teratoma Induction
3.2.1 Hind Limb Irradiation (Day 2)
3.2.2 Cardiotoxin Injury (Day 1)
3.2.3 Cell Transplantation (Day 0)
3.3 Teratoma Cells Isolation
3.4 Purification of Teratoma-Derived Skeletal Myogenic Progenitors
3.5 Transplantation of Teratoma-Derived Skeletal Myogenic Progenitors
3.6 Engraft Analysis
3.6.1 TA Muscle Harvest
3.6.2 Engraftment Quantification
4 Notes
References
Part II: Animal Models for Muscle Stem Cells & Regeneration
Chapter 14: Techniques for Injury, Cell Transplantation, and Histological Analysis in Skeletal Muscle
1 Introduction
2 Materials
2.1 Intramuscular Injection of Chemical Solutions for Inducing Muscle Regeneration
2.2 Intramuscular Transplantation of Cultured Myoblasts to Immunodeficient Mice
2.3 Isolation and Freezing of Muscle Samples
2.4 Sectioning and Staining of Muscle Samples
3 Methods
3.1 Intramuscular Injection of Chemical Solutions for the Induction of Muscle Regeneration
3.2 Intramuscular Transplantation of Cultured Myoblasts into TA Muscle of mdx Mice
3.3 Isolation and Freezing of the Muscle Samples
3.4 Cryosectioning and Staining of Muscle Samples
3.4.1 Cryosectioning
3.4.2 H&E Staining
3.4.3 Immunohistochemistry for dMyHC and Dystrophin
4 Notes
References
Chapter 15: Murine Models of Tenotomy-Induced Mechanical Overloading and Tail-Suspension-Induced Mechanical Unloading
1 Introduction
2 Materials
2.1 Tenotomy Model
2.2 Tail Suspension Model
3 Methods
3.1 Surgical Operation for Tenotomy
3.2 Tail Suspension Method
4 Notes
References
Chapter 16: Skeletal Muscle Denervation: Sciatic and Tibial Nerve Transection Technique
1 Introduction
2 Materials
2.1 Tools and Equipment
2.2 Animals
3 Methods
3.1 Sciatic Nerve Transection
3.2 Tibial Nerve Transection
4 Advanced Protocols
4.1 Muscle Atrophy
4.2 Reinnervation
4.3 Experimental Control of Denervation Study
4.4 Advanced Denervation Study
4.5 Partial Denervation
4.6 Hindlimb Unloading with Denervation
References
Chapter 17: Skeletal Muscle Regeneration in Zebrafish
1 Introduction
2 Materials
3 Methods
3.1 Preparation
3.2 Mounting Zebrafish Embryos/Larvae
3.3 Needlestick Muscle Injury
3.4 Laser-Induced Muscle Injury
3.5 Tracking Zebrafish Skeletal Muscle Regeneration
4 Notes
References!
Chapter 18: Methods to Monitor Circadian Clock Function in Skeletal Muscle
1 Introduction
2 Materials
2.1 Reagents
2.1.1 Mice
2.1.2 Luciferase Explant Media
2.1.3 2xDMEM Buffer Stock
2.1.4 1X Fresh Explant Medium
2.2 Supplies and Equipment
3 Methods
3.1 Preparation of Per2::Luc Muscle Explants for Bioluminescence Activity Recording
3.2 Preparation of Per2::Luc Primary Myoblasts for Bioluminescence Recording
3.3 LumiCycle 96 Bioluminescence Recording of Per2::Luc Muscle Explant or Myoblasts
3.4 Bioluminescence Data Analysis of Muscle Explant and Myoblast
4 Notes
References
Chapter 19: Visualizing MyoD Oscillations in Muscle Stem Cells
1 Introduction
2 Materials
2.1 MyoD-Luciferase Knock-In Reporter Mouse
2.2 Bioluminescence Imaging System, Image Processing, and Analysis
2.2.1 Image Acquisition
2.2.2 Computer and Monitor
2.2.3 Image Analysis
2.3 Other Materials
2.3.1 General Equipment and Solutions
2.3.2 Media and Reagents for Isolation of Single Muscle Stem Cells
2.3.3 Media and Solutions for Single Myofiber and Muscle Biopsies
3 Methods
3.1 Bioluminescence Imaging
3.2 Image Processing and Quantification
3.3 Preparation of Dissociated Muscle Stem Cells for Bioluminescence Imaging
3.4 Preparation of Myofiber-Associated Muscle Stem Cells for Bioluminescence Imaging
3.5 Preparation of Muscle Biopsies for Bioluminescence Imaging
4 Notes
References
Chapter 20: In Vivo Modeling of Skeletal Muscle Diseases Using the CRISPR/Cas9 System in Rats
1 Introduction
2 Materials
2.1 CRISPR/Cas9 Reagents
2.2 Embryo Collection
2.3 Injection of Cas9/sgRNA into Embryos
2.4 Transfer of Embryos to Pseudo-Pregnant Rats
2.5 Genotype PCR
3 Methods
3.1 Tips for Designing sgRNAs and Homologous Recombination Vector
3.2 Preparation of the Embryos from Female Rats
3.3 Microinjection of Cas9 and sgRNAs into the Embryos
3.4 Transfer of Embryos into Pseudo-Pregnant Rats
3.5 Genotyping and Confirmation of Mutation Pattern in F0 Pups
4 Notes
References
Chapter 21: In Vivo Investigation of Gene Function in Muscle Stem Cells by CRISPR/Cas9-Mediated Genome Editing
1 Introduction
2 Materials
2.1 Plasmid Construction
2.2 Testing Editing Efficiency Using SURVEYOR Nuclease Assay
2.3 AAV9 Virus Production, Purification, and Titration
2.4 AAV9 Virus Administration
3 Methods
3.1 sgRNA Selection
3.1.1 sgRNA Design
3.1.2 Testing the Editing Efficiency Using SURVEYOR Nuclease Assay
3.2 Construction and in Vitro Validation of AAV9-Dual sgRNA Vector
3.2.1 Construction of AAV9-Dual sgRNA Backbone
3.2.2 In Vitro Validation of the AAV9-Dual sgRNA Vector
3.3 AAV9 Virus Production, Purification, and Titration
3.3.1 AAV9 Virus Production and Purification
3.3.2 AAV9 Virus Titration
3.4 AAV9 Virus Administration and Satellite Cell (SC) Isolation
4 Notes
References
Chapter 22: Exons 45-55 Skipping Using Antisense Oligonucleotides in Immortalized Human DMD Muscle Cells
1 Introduction
2 Materials
2.1 Immortalized Muscle Cell Culture
2.2 Transfecting AOs
2.3 RT-PCR and Exon Skipping Analysis
2.4 Protein Extraction
2.5 Western Blotting and Dystrophin Expression Quantification
3 Methods
3.1 Immortalized Muscle Cell Culture
3.2 RT-PCR and Exon Skipping Analysis
3.3 Protein Extraction
3.4 Western Blotting and Dystrophin Rescue Quantification
4 Notes
References
Chapter 23: In Vivo Evaluation of Exon 51 Skipping in hDMD/Dmd-null Mice
1 Introduction
2 Materials
2.1 Design of Antisense Morpholinos
2.2 Intramuscular and Intravenous Injection of Antisense Morpholinos in Mice
2.3 Muscle Sampling
2.4 RT-PCR and Direct Sequencing of PCR Product
3 Methods
3.1 Design of Antisense Morpholinos
3.2 Intramuscular Injection of Antisense Morpholinos in Mice
3.3 Intravenous Injection of Antisense Morpholinos in Mice
3.4 Muscle Sampling
3.5 RT-PCR and Direct Sequencing of PCR Product
4 Notes
References
Part III: Bioinformatics & Imaging Analysis for Muscle Stem Cells
Chapter 24: Functional Analysis of MicroRNAs in Skeletal Muscle
1 Introduction
2 Materials
2.1 Measurement of Mature miRNAs by PCR
2.2 Measurement of Pri-miRNAs and Pre-miRNAs
2.3 miRNA-Target mRNA Interaction
2.4 Manipulation of miRNA Expression: Overexpression of miRNAs
2.5 Manipulation of miRNA Expression: Knockdown of miRNAs
3 Methods
3.1 Quantification of Mature miRNAs by Real-Time PCR
3.2 Quantification of Pri- and Pre-miRNAs by Semi-quantitative RT-PCR
3.3 miRNA-Target mRNA Interaction: Target Prediction Using the Online Database
3.4 miRNA-Target mRNA Interaction: Reporter Assay
3.5 Manipulation of miRNA Expression: Overexpression of miRNAs
3.6 Manipulation of miRNA Expression: Knockdown of miRNAs
4 Notes
References
Chapter 25: Targeted Lipidomics Analysis of Adipose and Skeletal Muscle Tissues by Multiple Reaction Monitoring Profiling
1 Introduction
2 Materials and Instrumentation
2.1 Samples
2.2 Materials
2.3 Equipment
2.4 Software
3 Methods
3.1 Lipid Extraction
3.2 Targeted Lipid Exploratory Analysis by MRM Profiling Methods
3.3 Instrument and Pump Settings
3.4 Quality Control (QC) and Blank Samples
3.5 Data Acquisition
3.6 Data Analysis
3.7 Comments on Biological Interpretation of MRM Lipid Profiling
4 Notes
References
Chapter 26: Single-Cell Transcriptomic Analysis of Mononuclear Cell Populations in Skeletal Muscle
1 Introduction
2 Materials
2.1 Cell Preparation Reagents
2.2 Cell Preparation Reagent Setup
2.3 Cell Preparation Equipment
2.4 Library Preparation Reagents
2.5 Library Preparation Reagent Setup
2.6 Library Preparation Equipment
2.7 Software for Data Analysis
3 Methods
3.1 Cell Preparation
3.2 Cell Sorting
3.3 Processing of Sorted Cells
3.4 GEM Generation and Barcoding
3.5 Post GEM-RT Cleanup and cDNA Amplification
3.6 3′ Gene Expression Library Construction
3.7 Sequencing
3.8 Basic Data Analysis
3.9 Ligand-Receptor Pairs
3.10 RNA Velocity
4 Notes
References
Chapter 27: Assay for Transposase-Accessible Chromatin Using Sequencing of Freshly Isolated Muscle Stem Cells
1 Introduction
2 Materials
3 Methods
3.1 Brief Description of the MuSC Isolation
3.2 Tagmentation
3.3 First PCR Amplification
3.4 Side qPCR Reaction
3.5 Second PCR Amplification
3.6 Double-Sided SPRI Bead Clean-up
3.7 Quality Assessment
3.8 Sequencing
3.9 Brief Description of the Bioinformatical Analysis
4 Notes
References
Chapter 28: Efficient Genome-Wide Chromatin Profiling by CUT&RUN with Low Numbers of Muscle Stem Cells
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffer Solutions
2.3 Equipment
2.4 Software
3 Methods
3.1 Preparation of ConA Beads
3.2 Binding of MuSCs to Beads
3.3 Primary Antibody Incubation
3.4 Secondary Antibody Incubation (Optional)
3.5 Binding of MNase Fusion Protein
3.6 Targeted DNA Digestion and Chromatin Release
3.7 DNA Extraction
3.8 Library Preparation and Sequencing
3.9 Data Processing and Analysis
4 Notes
References
Chapter 29: Epitranscriptome Mapping of N6-Methyladenosine Using m6A Immunoprecipitation with High Throughput Sequencing in Sk...
1 Introduction
2 Materials
2.1 RNA Fragmentation
2.2 RNA Immunoprecipitation, Elution, and Purification
2.3 qPCR (Optional)
2.4 Others
3 Methods
3.1 RNA Fragmentation (Approximately 3 Hours)
3.2 Formulation of Immunocomplex Between Free Antibody and Fragmented RNAs (Approx. 3 Hours)
3.3 Preparation of Pre-Cleared Magnetic Beads for Immunoprecipitation (Approx. 3 Hours)
3.4 Immunoprecipitation (Approx. 4 Hours)
3.5 Wash (Approx. 1 Hour)
3.6 Elution (Approx. 2.5 Hours)
3.7 Purification of Eluted m6A Marked RNA Fragments (Approx. 1 Hour)
3.8 Sequence of m6A IP RNA
3.8.1 m6A MeRIP Validation by qPCR (Optional)
3.8.2 MeRIP-Seq (Library Preparation)
4 Notes
References
Chapter 30: Visualization of RNA Transcripts in Muscle Stem Cells Using Single-Molecule Fluorescence In Situ Hybridization
1 Introduction
2 Materials
3 Methods
3.1 Preparation of the Confocal Dish (Day 1 and Day 2)
3.2 QSC Isolation Using FACS (Day 2)
3.3 SC Plating, Fixation, and Permeabilization (Day 2)
3.4 Wash and Hybridization (Day 3)
3.5 Wash and Mounting (Day 4)
4 Note
References
Chapter 31: Tissue Clearing and Confocal Microscopic Imaging for Skeletal Muscle
1 Introduction
2 Materials and Preparation
2.1 Tissue Dissection
2.2 Tissue Clearing
2.3 Embedding
2.4 Imaging
3 Methods
3.1 Perfusion Fixation and Muscle Dissection (Fig. 1)
3.2 Muscle Tissue Clearing
3.3 Embedding (Fig. 2)
3.4 Key Parameters for Image Acquisition
3.5 General Protocol for Image Acquisition (Fig. 3)
4 Notes
References
Chapter 32: Three-Dimensional Imaging Analysis for Skeletal Muscle
1 Introduction
2 Materials
2.1 Confocal Microscopic Imaging Files (e.g., Nikon A1R FLIM and FCS Confocal Microscope)
2.2 Image Analysis Software
3 Methods (Fig. 2)
3.1 Image Preparation
3.2 Cropping
3.3 Segmentation Training in Ilastik
3.4 Segmentation
3.5 3-D Rendering on Imaris
4 Notes
References
Index