Bone Marrow Environment 1st Edition by Marion Espéli, Karl Balabanian ISBN 107161424X 9781071614242

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ISBN 10: 107161424X
ISBN 13: 9781071614242 
Author: Marion Espéli, Karl Balabanian

Bone Marrow Environment 1st Edition Table of contents:

Part I: Cell Culture and In Vitro Functionnal Assays
Chapter 1: Culture, Expansion and Differentiation of Human Bone Marrow Stromal Cells
1 Introduction
2 Materials
2.1 Culture Media
2.2 Flow Cytometry
2.3 Histology, Dyes and Staining
3 Methods
3.1 Isolation of Human BM-MSC
3.1.1 Isolation of BM-MSC from Bone Fragments
3.1.2 Isolation of MSC from BM Aspirates
3.1.3 Isolation of Both BM-MSC and Hematopoietic Cells
3.2 Amplification and Banking of BM-MSC
3.3 Evaluation of MSC Clonogenicity
3.4 Phenotypical Characterization of MSC Population
3.5 Evaluation of MSC Expansion Capacities
3.6 Osteogenic Differentiation
3.7 Adipogenic Differentiation
3.8 Chondrocyte Differentiation
4 Notes
References
Chapter 2: Differentiation and Phenotyping of Murine Osteoclasts from Bone Marrow Progenitors, Monocytes, and Dendritic Cells
1 Introduction
2 Materials
2.1 Isolation of BM Cells
2.2 Cell Culture (Necessarily Sterile)
2.3 Magnetic Separation (See Note 4)
2.4 Phenotypic Analysis
3 Methods
3.1 Collection of Murine BM Cells
3.2 Differentiation of OCLs from Total BM Cells
3.3 OCL Differentiation from CD11b+Monocytic Cells (See Note 4)
3.4 OCL Differentiation from BM-Derived CD11c+ DCs
3.5 Validating the OCL Phenotype (See Note 17)
3.5.1 TRAcP Staining
3.5.2 Detachment of OCLs (See Notes 20-22)
3.5.3 Resorption Assays
3.5.4 Analysis of Multinucleation on OCLs
4 Notes
References
Chapter 3: Culture, Expansion and Differentiation of Mouse Bone-Derived Mesenchymal Stromal Cells
1 Introduction
2 Materials
2.1 Plastic and Equipment
2.2 Reagents
3 Methods
3.1 Isolation of Bone-Derived MSCs
3.1.1 BM Flush
3.1.2 Digestion of Compact Bones
3.2 Culture and Expansion of Bone-Derived MSCs
3.2.1 Cell Counting and Seeding
3.2.2 Cell Passaging and Splitting
3.3 Osteoblastic Differentiation
3.3.1 Osteoblastic Differentiation Culture
3.3.2 Alizarin Red S Staining and Quantification
4 Notes
References
Chapter 4: Feeder-Free Differentiation Assay for Mouse Hematopoietic Stem and Progenitor Cells
1 Introduction
2 Materials
2.1 Plastic and Equipment
2.2 Reagents
2.3 Antibodies and Dilutions
3 Methods
3.1 Marrow Sample Isolation
3.2 Depletion of Lineage-Positive Cells
3.3 Flow Cytometry Staining and Sorting
3.4 In Vitro HSPC Differentiation
3.5 Flow Cytometric Analyses
3.6 Additional Analyses
4 Notes
References
Chapter 5: In Vitro Analysis of Energy Metabolism in Bone-Marrow Mesenchymal Stromal Cells
1 Introduction
2 Materials
2.1 Bone Marrow MSCs
2.2 SeaHorse Experiment
3 Methods
3.1 Day Prior to Assay: Cartridge Calibration and Cell Plate Preparation
3.2 Day Prior to Assay: Template Preparation on Seahorse Wave Acquisition Software
3.3 Day of Assay: Cartridge Calibration and Cell Plate Preparation
3.4 Day of Assay: Cartridge Loading
3.5 Day of Assay: Run the XFe Test
3.6 Results Analysis
3.6.1 Mitochondrial Respiration Determination
3.6.2 Glycolytic Function Determination
3.6.3 Complementary Energetic Function Determination
4 Notes
References
Part II: Flow Cytometry-Based Analysis of the Bone Marrow Cellular Compartments
Chapter 6: Flow Cytometry Analysis of Mouse Hematopoietic Stem and Multipotent Progenitor Cells
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 BM Cell Recovery
3.2 Cell Staining
3.3 HSPC Characterization
4 Notes
References
Chapter 7: Flow Cytometry-Based Analysis of the Mouse Bone Marrow Stromal and Perivascular Compartment
1 Introduction
2 Materials
2.1 Cell Preparation
2.2 Cell Sorting and Single-Cell RNA-Seq Analysis
2.3 Colony-Forming Assay, Subsequent Subcloning and In Vitro Trilineage Differentiation
3 Methods
3.1 Cell Preparation
3.2 Cell Sorting and Single-Cell RNA-Seq Analysis of FACS-Isolated Cells
3.3 Colony-Forming Assay, Subcloning, and In Vitro Trilineage Differentiation
4 Notes
References
Chapter 8: Immunophenotyping of the Medullary B Cell Compartment In Mouse Models
1 Introduction
2 Materials
2.1 Material and Tools
2.2 Buffers and Media
2.3 Equipment
3 Methods
3.1 Mouse Experimentation
3.2 Cell Preparation
3.3 Flow Cytometry Staining
3.4 Flow Cytometry Analysis
4 Notes
References
Chapter 9: Metabolic Analysis of Mouse Hematopoietic Stem and Progenitor Cells
1 Introduction
2 Materials
2.1 Cell Preparation
2.2 Cell Surface Staining
2.3 Assessment of ROS Levels
2.4 Intracellular Staining for Activated Stress Signaling Pathways
3 Methods
3.1 Cell Preparation
3.2 Cell Surface Staining
3.3 Assessment of Intracellular and Mitochondrial ROS
3.4 Assessment of Activated Stress Signaling Pathways
4 Notes
References
Part III: Imaging of the Bone Marrow
Chapter 10: Multicolor Immunofluorescence Staining of Paraffin-Embedded Human Bone Marrow Sections
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 11: 3D Microscopy of Murine Bone Marrow Hematopoietic Tissues
1 Introduction
2 Materials
2.1 Fixation Buffer
2.2 Cryopreservation and Sectioning
2.3 Immunohistology
2.4 Microscopy and Image Analysis
3 Methods
3.1 BM Harvesting and Tissue Processing
3.2 Generation of Thick Slices of BM Tissues
3.3 Immunostaining and Tissue Clearing
3.4 Deep Tissue Imaging
4 Notes
References
Chapter 12: Ex Vivo Whole-Mount Imaging of Leukocyte Migration to the Bone Marrow
1 Introduction
2 Materials
2.1 Harvest and Staining of Cells
2.2 Adoptive Transfer, Blood Vasculature Staining, and Bone Harvest
2.3 Preparation of Bone Marrow
3 Methods
3.1 Extraction and Staining of Cells for Injection
3.2 Adoptive Transfer, Blood Vasculature Staining, and Bone Harvest
3.3 Preparation of Bones for Whole-Mount Imaging and Analysis
4 Notes
References
Chapter 13: Intrafemoral Delivery of Hematopoietic Progenitors
1 Introduction
2 Materials
3 Methods
3.1 Cell Sorting
3.2 Intrafemoral Transfer
3.3 Flow Cytometry Analysis
4 Notes
References
Chapter 14: Imaging of Bone Marrow Plasma Cells and of Their Niches
1 Introduction
2 Materials
2.1 Animals
2.2 Buffers
2.3 Single-Cell Suspensions (in Case of Cell Transfer/BM Chimera)
2.4 B cell isolation (in Case of Adoptive Transfer with Subsequent Immunization)
2.5 Cell Transfer and Immunization
2.6 Drugs
2.7 Devices and Tools
2.8 Imaging Equipment
2.9 Software
2.10 Additional Material
3 Methods
3.1 Bone Marrow Chimera
3.2 Cell Isolation and Adoptive Transfer
3.3 Immunization (in Case of Cell Transfer)
3.4 Surgical Procedure
3.5 Imaging
4 Notes
References
Chapter 15: Intravital Imaging of Bone Marrow Microenvironment in the Mouse Calvaria and Tibia
1 Introduction
2 Materials & Equipment
2.1 Mouse
2.2 Anesthesia
2.3 Hair Removal for Skull Imaging
2.4 Hair Removal for Tibial Imaging
2.5 Intravenous Injection
2.6 Skin Incision of the Calvarium
2.7 Skin, Muscle, and Tibia Incision
2.8 Calvarial Bone Marrow Stage
2.9 In Vivo Multiphoton Imaging
3 Methods
3.1 Preparation of Coverslip Holder for Skull Bone Marrow Imaging
3.2 Mouse Anesthesia for Skull Bone Marrow Imaging
3.3 Scalp Hair Removal for Skull Bone Marrow Imaging
3.4 Scalp Skin Incision for Skull Bone Marrow Imaging
3.5 Blood Flow Labelling for Skull Bone Marrow Imaging
3.6 Positioning Mouse Skull onto Customized Stereotactic Frame Stage for Skull Bone Marrow Imaging
3.7 Imaging Setup for Skull Bone Marrow Imaging
3.8 Surgical Stage Preparation for Tibial Bone Marrow Imaging
3.9 Mouse Anesthesia for Tibial Bone Marrow Imaging
3.10 Thigh Hair Removal for Tibial Bone Marrow Imaging
3.11 Tibial Surgery for Tibial Bone Marrow Imaging
3.11.1 Mouse Placement to Surgical Stage
3.11.2 Skin Incision and Tissue Removal
3.11.3 Tibia Shaving
3.11.4 Blood Flow Labelling
3.11.5 Coverslip Positioning
3.12 Imaging Setup for Tibial Bone Marrow Imaging
4 Notes
References
Chapter 16: Intravital Imaging of Bone Marrow Niches
1 Introduction
2 Materials
2.1 Mice
2.2 Surgery and Intravital Microscopy Reagents, Supplies, and Equipment
2.3 Image Analysis
3 Methods
3.1 Microscope Considerations for In Vivo Imaging
3.2 Surgery
3.3 Mounting the Mouse onto the Stage
3.4 Image Acquisition Setup
3.5 Recovery of the Mouse for Reimaging
3.6 Image Processing and Analysis
3.7 Application of the Protocol for Measuring Vascular Permeability
4 Notes
References
Part IV: In Vivo and Ex Vivo Modelling of the Bone Marrow Ecosystem
Chapter 17: Modeling Human Fetal Hematopoiesis in Humanized Mice
1 Introduction
2 Materials
2.1 Cell Preparation and Culture Plates
2.2 Magnetic Cell Separation Kits
2.3 Buffers and Media
2.4 Handling of Immunodeficient Mice
2.5 Antibody Staining
2.6 Data Collection and Analysis
3 Methods
3.1 Purification and Storage of CD34+ HPC from Umbilical Cord Blood (UCB) (See Note 2)
3.2 CD34+ Cell Activation and Xenotransplantation
3.3 Isolation of Human Cells from the BM of Xenografted Mice
3.4 Antibody Staining
3.5 Flow Cytometry Gating Strategy (Fig. 1)
4 Notes
References
Chapter 18: Engraftment of Human Hematopoietic Cells in Biomaterials Implanted in Immunodeficient Mouse Models
1 Introduction
2 Materials
2.1 Human Sample Preparation
2.2 Seeding of Collagen-Based Scaffolds
2.3 Scaffold Implantation
2.4 Retrieval of Cells After Xenotransplantation
2.5 Solutions and Culture Medium
3 Methods
3.1 hMSC and HUVEC Culture
3.2 Bioengineering Collagen-Based 3D Scaffolds with Human HSPCs and Stromal Cells
3.2.1 Prepare hMSCs and HUVECs
3.2.2 Preparation of Collagen-Based Scaffolds
3.2.3 Seeding of Collagen-Based Scaffolds with Stromal Cells
3.2.4 Isolation of Human HSPCs
3.2.5 Seeding of Collagen-Based Scaffolds with Human HSPCs
3.3 Surgical Implantation of 3D Scaffolds in Immunodeficient Mice
3.3.1 Animal Preparation
3.3.2 Surgical Implantation
3.4 Mouse Euthanasia, Tissue Retrieval, and Tissue Digestion
3.4.1 Mouse Euthanasia
3.4.2 Tissue Retrieval
3.4.3 Tissue Digestion
3.4.4 Sample Processing for Direct Flow Cytometry
4 Conclusion
5 Notes
References
Chapter 19: 3D Engineering of Human Hematopoietic Niches in Perfusion Bioreactor
1 Introduction
2 Materials
2.1 Isolation of Primary Human Bone Marrow MSCs and Cord Blood CD34+ HSPCs
2.2 3D Perfusion Bioreactor
2.3 Preparation for Flow Cytometry and Histology
3 Methods
3.1 Primary Human Bone Marrow MSC Isolation
3.2 Cord Blood CD34+ HSPC Isolation
3.3 Establishment of an Osteoblastic Niche in the 3D Perfusion Bioreactor
3.4 Seeding and Coculture of Cord Blood CD34+ HSPCs on Engineered Osteoblastic Niches
3.5 Sample Harvesting for Flow Cytometry
3.6 Sample Harvesting for Histology
4 Notes
References
Chapter 20: Manufacturing a Bone Marrow-On-A-Chip Using Maskless Photolithography
1 Introduction
2 Materials
2.1 Microfabrication
2.2 Hydrogels
2.3 Cells and Culture Media
3 Methods
3.1 Virtual Mask Design
3.2 Master Mold Fabrication
3.3 Microfluidic Chip Molding and Assembling
3.4 Hydrogel Loading with Cells
4 Notes
References
Part V: Sequencing-Based Analysis of Cell Heterogeneity
Chapter 21: In Vivo Tracking of Hematopoietic Stem and Progenitor Cell Ontogeny by Cellular Barcoding
1 Introduction
2 Materials
2.1 Common Reagents
2.2 Barcode Virus Production
2.3 Isolation of HSPCs
2.4 Transduction of HSPCs with Barcode Virus
2.5 Transplantation of Transduced HSPCs
2.6 Isolation of Target Populations
2.7 PCR Amplification of Barcode DNA
2.8 Sequencing
2.9 Data Processing and Analysis
3 Methods
3.1 Barcode Virus Production
3.2 Isolation of HSPCs
3.3 Transduction of HSPCs with Barcode Virus and Transplantation
3.4 Isolation of Target Populations
3.5 PCR Amplification of Barcode DNA
3.6 Sequencing
3.7 Data Processing and Analysis
4 Notes
References
Chapter 22: Single-Cell Analysis of Hematopoietic Stem Cells
1 Introduction
2 Materials
2.1 Bone Marrow Harvest
2.2 Antibodies
2.3 scRNA-Seq
2.4 Oligonucleotides
3 Methods
3.1 Sample Preparation
3.1.1 Harvest Bone Marrow
3.1.2 Single Antibody Staining Controls
3.1.3 Lineage Depletion
3.1.4 Specific Antibody Staining of Samples
3.2 Droplet-Based scRNA-Seq Using the 10x Genomics Platform
3.3 Plate Based scRNA-Seq Using Molecular Crowding Smart-Seq2 (mcSmart-Seq2)
3.3.1 Single-Cell Sorting
3.3.2 Cell Lysis and Hybridization
3.3.3 Reverse Transcription
3.3.4 PCR Preamplification
3.3.5 PCR Purification
3.3.6 Quality Check the cDNA Library
3.3.7 cDNA Quantification
3.3.8 Library Preparation
3.3.9 Library Pooling and Clean up
3.3.10 Quality Check and Quantification of the Indexed Library
3.3.11 Pooling of Multiple Samples for Next Generation Sequencing
3.3.12 Sequencing
4 Notes
References
Index

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