Hematopoietic Stem Cells: Methods and Protocols

Description

This detailed volume collects updates on the technical advances in hematopoietic stem cell research and incorporates new techniques focused on the molecular/genetic, cellular, and whole organism levels. Exploring methods that apply stress to hematopoiesis, the book also contains chapters focused on better understanding the role of hematopoietic niches and their cellular components, as well as in vivo models that test and quantitate stem cell function and are key to further development of therapeutic applications. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and up-to-date, Hematopoietic Stem Cells: Methods and Protocols serves as a valued addition to laboratories focused on understanding hematopoietic stem cell biology and the therapeutic advances that can be derived from it. Preface Contents Contributors Part I: Overview Chapter 1: The Factory of Blood Production: Hematopoietic Stem Cells 1 Introduction 2 Molecular Modification and Evaluation 3 Cellular Isolation and Characterization 4 The Hematopoietic Niche and Accessory Cells 5 Transplantation and In Vivo Models 6 Conclusions Part II: Molecular Modification and Evaluation Chapter 2: Detection of DNA Damage in Hematopoietic Stem Cells 1 Introduction 2 Materials 2.1 Obtaining Hematopoietic Stem Cells 2.2 Generating Control Cells 2.3 Sample and Agarose Preparation 2.4 Sample Lysis and Agarose Preparation 2.4.1 Comet Assay 2.4.2 Halo Assay 2.5 Sample Staining and Imaging 2.6 DNA Damage Quantification 3 Methods 3.1 Obtaining Hematopoietic Stem Cells 3.2 Generating Control Cells 3.3 Sample and Agarose Preparation 3.4 Sample Lysis and DNA Separation 3.4.1 Comet Assay 3.4.2 Halo Assay 3.5 Sample Staining and Imaging 3.6 DNA Damage Quantification 3.6.1 Evaluation of Comet Data 3.6.2 Evaluation of Halo Data 3.7 Visualization of Data 4 Notes References Chapter 3: Evaluating Histone Acetylation in Mouse Hematopoietic Stem and Progenitor Cells Using Chromatin Immunoprecipitation 1 Introduction 2 Materials 3 Methods 3.1 Bone Marrow Isolation 3.2 Mouse Hematopoietic Progenitor Cell Isolation 3.3 In Vivo Crosslink and Lysis 3.4 Sonication to Shear DNA 3.5 Elution of Protein/DNA Complexes 3.6 Reverse Cross-Links of Protein/DNA Complexes to Free DNA and DNA Purification 3.7 Design Primers for the Genes and Perform Real-Time Quantitative PCR 4 Notes References Chapter 4: CRISPR Gene Editing of Hematopoietic Stem and Progenitor Cells 1 Introduction 1.1 Overview 1.2 HSPCs 1.3 CRISPR Nucleases 1.4 Guide RNA 1.5 Targeted Genomic Loci for Gene Editing 1.6 DNA Repair 1.6.1 Nonhomologous DNA Repair 1.6.2 Homologous DNA Repair 1.7 Analysis of Gene Editing 1.7.1 Analysis of On-Target Gene Editing 1.7.2 Analysis of Off-Target CRISPR-Cas Activity 2 Materials 2.1 HSPC Culture 2.2 Gene Editing Reagents and Equipment 2.3 Analysis Reagents 3 Methods 3.1 HSPC Priming in Culture 3.2 Electroporation and Transduction 3.3 Analysis of CRISPR Gene Edited HSPC 3.4 Extraction of gDNA from Bulk HSPC and Peripheral Blood Following Transplantation 3.5 Extraction of gDNA from Colony-Forming Cells 3.6 PCR Amplification and Sequencing of Target Genomic Loci of Interest 3.7 Analysis of Off-Target Effects 4 Notes 4.1 Controls 4.2 HSPC Media Selection 4.3 gRNA Search 4.4 Analysis of HSPC Fitness 4.4.1 Flow Cytometry 4.4.2 Colony-Forming Assay 4.4.3 In Vivo Transplantation 4.5 Electroporation 4.6 PCR Amplification and Sequencing of Genomic Target 4.7 CFU Analysis References Chapter 5: Lentiviral Transduction of Nonhuman Primate Hematopoietic Stem and Progenitor Cells 1 Introduction 2 Materials 2.1 Animals 2.2 Lentivirus Production and Titration Reagents and Supplies 2.3 CD34 Immunoadsorption Reagents and Supplies 2.4 Transduction Reagents and Supplies 3 Methods 3.1 Lentivirus Production via Transient Transfection 3.2 Vector Titration Using HeLa Cells 3.3 Purification of CD34+ HSPCs 3.4 Lentiviral Transduction of RM CD34+ HSPCs 3.5 Summary 4 Notes References Part III: Cellular Isolation and Characterization Chapter 6: Identification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells 1 Introduction 2 Materials 2.1 Reagents 2.2 Equipment 3 Methods 3.1 Sample Collection and Processing 3.2 Preparation of Samples for Flow Cytometry 3.3 Analysis of Samples on Flow-Cytometer 3.4 Flow-Cytometric Data Analysis - Comprehensive/High Resolution (See Fig. 1 and Table 3) 3.5 Flow-Cytometric Data Analysis - Short/Low Resolution (See Fig. 2 and Table 4) 4 Notes References Chapter 7: Isolation and Characterization of Fetal Liver Hematopoietic Stem Cells 1 Introduction 2 Materials 2.1 Mice 2.2 Reagents and Supplies 2.2.1 Supplies 2.2.2 0.5 M EDTA 2.2.3 Staining Media (5 mM EDTA +1x DPBS -/- + 2% FBS) 2.2.4 10x ACK (Ammonium-Chloride-Potassium) Lysis Buffer 2.2.5 Digestion Buffer 3 Methods 3.1 Timed Mating 3.2 Embryo Isolation 3.3 Fetal Liver Isolation and Processing 3.3.1 Manual Trituration 3.3.2 Enzymatic Digestion 3.4 HSC Enrichment Techniques 3.4.1 C-Kit Enrichment 3.5 Cell Surface Markers and Flow Cytometry for Fetal HSCs 3.5.1 Antibody Cocktail Preparation 3.5.2 Fetal HSC Gating Strategy 3.6 Fetal HSC Function in Transplantation 3.6.1 Recipients 3.6.2 Irradiation 3.6.3 Cell Numbers and Transplantation 4 Notes References Chapter 8: Utilizing CyTOF to Examine Hematopoietic Stem and Progenitor Phenotype 1 Introduction 2 Materials 2.1 Conjugation of Metal-Tagged Antibodies 2.2 Isolation of Lineage-Depleted Bone Marrow Cells 2.3 Reagents for Cell Surface and Intracellular Staining 3 Methods 3.1 Conjugation of Metal-Tagged Antibodies 3.2 Titration of Metal-Tagged Antibodies 3.3 Isolation of Lineage-Depleted Bone Marrow Cells 3.4 Staining Protocol for Cell Surface and Intracellular Antibodies 3.5 Data Analysis 4 Notes References Chapter 9: Hematopoietic Stem Cell Identification Postirradiation 1 Introduction 1.1 Radiation-Induced Marker Changes 1.2 Radiation-Induced Debris and Autofluorescence 1.3 Limiting Numbers of Bone Marrow Cells 2 Materials 3 Methods 3.1 Extraction of Multiple Bone Types 3.2 Crushing to Release Bone Marrow Cells 3.3 Surface Staining Bone Marrow Cells for Flow Cytometry 3.4 Intracellular Staining 3.5 Flow Cytometry Analysis of HSCs Postirradiation 4 Notes References Part IV: The Hematopoietic Niche and Accessory Cells Chapter 10: Intravital Microscopy for Hematopoietic Studies 1 Introduction 2 Materials 2.1 Mice 2.2 Reagents, Supplies, and Equipment 2.2.1 HSC Sort and Transplant 2.2.2 Surgery and Intravital Microscopy 2.2.3 Image Analysis 3 Methods 3.1 Bone Marrow Transplant 3.1.1 Conditioning of BM Recipients 3.1.2 HSC Preparation and Injection 3.2 Intravital Microscopy 3.2.1 Microscope Considerations for In Vivo Imaging 3.2.2 Surgery 3.2.3 Mounting the Mouse onto the Stage 3.2.4 Locating and Imaging HSCs 3.2.5 Recovery of the Mouse for Re-imaging 3.3 Image Processing and Analysis 4 Notes References Chapter 11: Laser Micromachining of Bone as a Tool for Studying Bone Marrow Biology 1 Introduction 2 Materials 2.1 Materials and Supplies for Installation of the Ablation Capability 2.1.1 Optical Components 2.1.2 Mechanical Components 2.2 Materials and Supplies for Laser Ablation 2.3 Materials for In Vivo Imaging 3 Methods 3.1 Installing a Laser Ablation Capability on Your Multiphoton Microscope 3.1.1 Installation of the Ablation Laser Unit 3.1.2 Installation of Flushing System 3.2 Laser Bone Thinning to Improve Image Quality 3.2.1 Perform Skin Flap Surgery 3.2.2 Perform In Vivo Multiphoton Microscopy 3.2.3 Perform Bone Thinning 3.3 Generation of Bone Defects 3.3.1 Perform Skin Flap Surgery, Multiphoton Microscopy, and Laser Injury 3.3.2 Survival Surgery 3.3.3 Follow-Up Imaging 4 Notes References Chapter 12: MSC and HSPC Coculture: Mimicking Ex Vivo Bone Marrow Niche 1 Introduction 2 Materials 3 Methods 3.1 MSC Isolation and Ex Vivo Expansion 3.1.1 Isolation of Mononuclear Cells 3.1.2 Enrichment of MSCs 3.1.3 In Vivo Expansion of MSCs 3.1.4 MSC Evaluation 3.2 Isolation of HSPC-Enriched Human CD34+ Cells 3.3 MSC and HSPC Coculture 4 Notes References Chapter 13: Isolation of Thymus Stromal Cells from Human and Murine Tissue 1 Introduction 2 Materials 2.1 Isolation of Murine Thymus 2.2 Dissociation of Murine Thymic Tissue 2.3 Magnetic Depletion of Hematopoietic Cells 2.4 Flow Cytometric Analysis of Murine Thymus Stromal Cells 2.5 Dissociation of Human Thymic Tissue 2.6 Flow Cytometric Analysis of Human Thymus Stromal Cells 3 Methods 3.1 Isolation of Murine Thymus 3.2 Dissociation of Murine Thymic Tissue 3.3 Magnetic Depletion of Hematopoietic Cells 3.4 Flow Cytometric Analysis of Murine Thymus Stromal Cells 3.5 Dissociation of Human Thymic Tissue 3.6 Flow Cytometric Analysis of Human Thymus Stromal Cells 4 Notes References Part V: Transplantation and In Vivo Models Chapter 14: Experimental Models of Mouse and Human Hematopoietic Stem Cell Transplantation 1 Introduction 2 General Transplant (Non-competitive) 2.1 Introduction 2.2 Materials 2.2.1 General 2.2.2 Infusion 2.2.3 PB Collection and Analysis 2.2.4 Phenotype Staining Reagents 2.3 Methods 2.3.1 Obtain Recipient Mice 2.3.2 Start Recipient Prophylaxis 2.3.3 Irradiate/Precondition Recipient Mice F1 Hybrid (C57BL/6 x BoyJ) 2.3.4 Donor BM Cell Harvest 2.3.5 Prepare Infusion Dilutions 2.3.6 Infusion 2.3.7 Post-transplant PB Collection 2.3.8 Staining for Flow Cytometry Analysis 2.3.9 Analysis by Flow Cytometry 2.4 Notes 3 Competitive Transplant 3.1 Introduction 3.2 Materials 3.2.1 General 3.2.2 Infusion 3.2.3 PB Collection and Analysis 3.2.4 Phenotype Staining Reagents 3.3 Methods 3.3.1 Obtain Recipient Mice 3.3.2 Start Recipient Prophylaxis 3.3.3 Irradiate/Precondition Recipient Mice F1 Hybrid (C57BL/6 x BoyJ) 3.3.4 Donor BM Cell Harvest 3.3.5 Prepare Infusion Dilutions 3.3.6 Infusion 3.3.7 Post-transplant PB Collection 3.3.8 Staining and Flow Cytometry Analysis 3.4 Notes 4 Xenograft Transplant 4.1 Introduction 4.2 Materials 4.2.1 General 4.2.2 Infusion 4.2.3 PB Collection and Analysis 4.2.4 Phenotype Staining Reagents/Materials 4.3 Methods 4.3.1 Obtain Recipient Mice 4.3.2 Start Recipient Prophylaxis 4.3.3 Irradiate/Precondition Recipient Mice NSG 4.3.4 Donor Cell Preparation 4.3.5 Prepare Infusion Dilutions 4.3.6 Infusion 4.3.7 Post-transplant PB Collection 4.3.8 Staining for Flow Cytometry Analysis 4.3.9 Analysis by Flow Cytometry 4.4 Notes References Chapter 15: Hematopoietic Stem and Progenitor Cell Identification and Transplantation in Zebrafish 1 Introduction 2 Materials 2.1 Zebrafish 2.1.1 Larval Transplantation Recipients 2.1.2 Larval Transplantation Donors 2.1.3 Adult Transplantation Recipients 2.1.4 Adult Transplantation Donors 2.2 Reagents and Supplies 2.3 Tools and Instruments 3 Methods 3.1 Donor Cell Harvest from Adult Zebrafish WKM 3.2 Donor Cell Harvest from Zebrafish Embryos and Larvae 3.3 FACS of Donor Cells for Transplantation 3.4 Larval Transplantation 3.4.1 Larval Transplantation: Prepare Recipient Embryos 3.4.2 Larval Transplantation: Cell Injection into Larval Recipients 3.4.3 Larval Transplantation: Post-transplant Care of Larval Recipients 3.5 Adult Transplantation 3.6 Measuring Engraftment in Transplant Recipients 3.6.1 Imaging-Based Engraftment Assessment 3.6.2 Flow Cytometry-Based Engraftment Assessment 4 Notes References Chapter 16: Establishing a Murine Model of the Hematopoietic Acute Radiation Syndrome 1 Introduction 2 Materials 2.1 Mice 2.2 Mouse Housing and Husbandry 2.3 Irradiation Materials 3 Methods 3.1 Mice and Husbandry 3.2 Irradiation 4 Notes References Chapter 17: Purinergic Signaling and Its Role in Mobilization of Bone Marrow Stem Cells 1 Introduction 2 Materials 2.1 Purinergic Receptor Activators and Inhibitors 2.2 Inhibitors of Ectonucleotidases 2.3 Other Crucial Proteins Involved in Purinergic Signaling and Its Biological Downstream Effects 2.4 Mobilization of Murine Stem Cells 2.4.1 Animals 2.4.2 Reagents 2.5 FACS Analysis of Stem Cells Mobilized into PB 2.5.1 Reagents 2.5.2 Equipment 2.6 Confocal Analysis of Membrane Lipid Rafts 2.6.1 Reagents 2.6.2 Equipment 2.6.3 Software 3 Methods 3.1 Application of Small Molecular Modulators of Purinergic Signaling in Stem Cell Mobilization Experiments 3.1.1 Modulators of Pannexin-1 Signaling 3.1.2 P2X7 and P2X4 Inhibitors 3.1.3 Modulators of Nlrp3 Activity 3.2 Analysis of Different Population of BM Mobilized Cells in Murine PB 3.2.1 Mobilization of Mice 3.2.2 Peripheral Blood Collection of Murine Cells and Red Blood Cell Lysis for FACS Analysis 3.2.3 Staining with Monoclonal Antibodies (mAb) 3.2.4 Flow Cytometry Analysis 3.3 Confocal Analysis of Membrane Lipid Raft Formation 4 Notes References Index