Pericytes: Methods and Protocols (Methods in Molecular Biology, 2235)

✍ Scribed by Bruno M. Péault (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 195
• Category: Library

✦ Synopsis

This volume explores the techniques used to study human and animal pericytes. Chapters in this volume cover a range of topics such as pericyte dissociation from tissues, and analysis by immunohistochemistry and flow cytometry; avian chimaeras to investigate the embryonic development of cell lineage; pericyte purification by fluorescence activated cell sorting and ensuing culture; tissue turnover and regeneration; and the dynamic study of pericyte involvement in cancer dissemination, in situ, and in culture. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical, Pericytes: Methods and Protocols is a valuable resource that helps researchers define the biologic identity, functions, and potentials ofa cell lineage of increasing interest and therapeutic significance.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Visualizing Pericyte Mimicry of Angiotropic Melanoma by Direct Labeling of the Angioarchitecture
1 Introduction
2 Materials
2.1 Reagents, General Supplies, and Equipment
2.2 Cells and Cell Culture Reagents
2.3 Experimental Animals and Reagents
2.4 Brain Tissue Processing
2.5 Fluorescence Imaging
3 Methods
3.1 Cell Culture
3.2 Stereotaxic Brain Injection
3.3 Jugular Vein Injections
3.4 Tissue Processing for Microscopy
3.5 Fluorescence Microscopy Imaging
4 Notes
References
Chapter 2: Transplantation of Purified Human Pericytes for Myocardial Repair in SCID-hu Mice
1 Introduction
2 Materials
2.1 Cell Purification/Culture Media and Solutions
2.2 Cell Cultureware
2.3 Animals
2.4 Animal Surgery Equipment and Supply
3 Methods
3.1 Human Tissue Biopsy Processing
3.2 Tissue Dissociation and Cell Isolation
3.3 Cell Sorting
3.4 Pericyte Culture
3.5 Preparation of Animals and Donor Cells
3.6 Anesthesia and Preparation of the Surgical Site
3.7 Infarct Induction and Cell Transplantation
4 Notes
References
Chapter 3: Assessment of Pericyte Phenotype by Flow Cytometry
1 Introduction
2 Pericyte Characterization by Flow Cytometry (FACS)
2.1 Material
2.1.1 Equipment
2.1.2 Solutions
2.1.3 Antibodies
3 Methods
3.1 Tissue Procurement and Preparation
3.2 Tissue Dissociation
3.2.1 Soft Tissues (Placenta, Kidney, Others)
3.2.2 Connective Soft Tissue (Adipose Tissue)
3.3 Staining
3.4 Flow Cytometry Presort Preparation
3.4.1 Instrument Details
3.4.2 Instrument Preparation
3.5 Cell Analysis and Sorting
4 Notes
References
Chapter 4: Characterization of Human Pericyte Phenotype by Immunohistochemistry
1 Introduction
2 Pericyte Characterization by Immunohistochemistry
2.1 Material
2.1.1 Equipment
2.1.2 Solutions
2.1.3 Staining Reagents
2.2 Antibodies
2.2.1 Primary Antibodies
2.2.2 Secondary Antibodies for Fluorescence Staining
2.2.3 Secondary Antibodies for Nonfluorescence Staining
3 Methods
3.1 Tissue Procurement and Preparation
3.1.1 OCT Inclusion
3.1.2 Sucrose-Gelatin Inclusion
3.2 Immunostaining of Tissue Sections
3.2.1 Fluorescence Double Staining
3.2.2 Nonfluorescence Double Staining
4 Notes
References
Chapter 5: A Metabolomics-Based Approach to Identify Lineage Guiding Molecules in Pericyte Cultures
1 Introduction
2 Materials
2.1 Pericyte Isolation
2.2 Cell Culture
2.3 Hydrogels
2.4 Metabolite Extraction
2.5 LC-MS
2.6 Cytotoxicity
3 Methods
3.1 Pericyte Isolation
3.2 Hydrogel Preparation and Cell Culture
3.3 Metabolite Extraction
3.4 Liquid Chromatography-Mass Spectrometry
3.5 Selecting Metabolites for Further Testing
3.6 Cytotoxicity
4 Notes
References
Chapter 6: Pericyte Ontogeny: The Use of Chimeras to Track a Cell Lineage of Diverse Germ Line Origins
1 Introduction
1.1 Brief History of the Mysterious Pericyte
1.2 An Adaptable Tool: Xenografting
1.3 Multiple Origins of Pericytes-Neural Crest
1.3.1 Abutting Pericyte Lineage Tracing
1.4 Multiple Origins of Pericytes-Mesoderm
1.4.1 Epicardium
1.4.2 Mesothelium
1.4.3 Sclerotome
2 Materials
2.1 Eggs
2.2 Incubators and Handling
2.3 Microdissection Tools
2.4 Other Equipment
2.5 Solutions
3 Methods
3.1 Host Egg Preparation
3.2 Host Microdissection
3.3 Graft Egg Preparation
3.4 Graft Microdissection
3.5 Harvesting, Fixation, and Processing for Embedding
3.6 Immunolabeling
4 Notes
5 Discussion
5.1 Fate-Mapping Can Further Understanding of the Pathophysiology of Vascular Malformations
5.2 Novel Cellular Contributions to Secretory Function
5.3 Pericytic Mimicry and Physiological Stem Cell Niches
References
Chapter 7: Imaging Pericytes and the Regulation of Cerebral Blood Flow
1 Introduction
1.1 Signals Generated by Neuronal Activity
1.2 Vascular Responses to Neuronal Activity
1.3 Ex Vivo Brain Slice Imaging
2 Protocol for Imaging Neurovascular Coupling in Ex Vivo Brain Slices
2.1 Summary
2.2 Slicing Solutions
2.3 Experimental Solutions
2.4 Dissection and Production of Brain Slices Using a Vibratome
2.5 Locating Vessels
2.6 Image Acquisition
2.7 Image Analysis
2.7.1 Registration
2.7.2 Measurement
3 In Vivo Imaging
3.1 Cranial Window Surgery
4 Medication for a 25-g Mouse
4.1 Dye Injection
4.2 Imaging
4.3 Analysis
5 Immunohistochemistry Experiments
6 Solutions
6.1 Phosphate-Buffered Saline (PBS) Solution, 0.1 M
6.2 Paraformaldehyde (PFA) Solution, 4% (w/v)
6.3 Blocking and Permeabilizing Solution (Block-Perm)
7 Methods
8 Conclusion
9 Notes
References
Chapter 8: Derivation of Pericytes from Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Human Pluripotent Cell Lines (hPSCs)
2.2 Culture Media
2.3 Enzymes and Solutions
2.4 Antibodies
2.5 Equipment
3 Methods
3.1 Differentiation of hPSCs
3.2 EB Dissociation
3.3 Magnetic Cell Separation
3.4 Expansion of Isolated CD146+ hPSC-Pericytes
4 Notes
References
Chapter 9: Assessing the Bone-Forming Potential of Pericytes
1 Introduction
2 Materials
2.1 Mouse Intramuscular Application
2.2 Mouse Calvarial Defect Application
2.3 Rat Spinal Fusion
3 Methods
3.1 Mouse Intramuscular Application
3.2 Mouse Calvarial Defect Application (Fig. 1)
3.3 Rat Spinal Fusion Application (Fig. 2)
3.4 Assessments of Bone Formation
4 Notes
References
Chapter 10: Manipulating Pericyte Function with MicroRNAs
1 Introduction
2 Materials
2.1 Cell Culture
2.2 miRNA Overexpression/Inhibition
2.3 EdU Cell Proliferation Assay
2.4 Transwell Migration Assay
2.5 Insert Preparation
2.6 Ligation, Transformation, and Transfection
2.7 Luciferase and β-Galactosidase Reactions
3 Methods
3.1 Transfection
3.2 Functional Assays
3.2.1 Edu Cell Proliferation Assay
3.2.2 Transwell Migration Assay
3.3 Cloning Protocol for miRNA Target Validation
3.3.1 Preparing Inserts for Ligation by PCR
3.3.2 Ligation and Transformation
3.3.3 CaCl2 Transfection
3.3.4 Luciferase Screen
3.3.5 β-Galactosidase Assay
4 Notes
References
Chapter 11: Isolation of Mammalian Mesoangioblasts: A Subset of Pericytes with Myogenic Potential
1 Introduction
2 Materials
2.1 Basic Materials
2.2 Media
3 Methods
3.1 Fluorescence-Activated Cell Sorter (FACS) Isolation of Adult Murine MABs
3.2 FACS Isolation of Adult Human-Equine-Canine MABs
3.3 FACS Isolation of Mouse and Human Cardiac MABs
3.4 Co-culture with C2C12
3.5 Spontaneous Differentiation
3.6 Smooth Muscle Differentiation
3.7 Osteogenic Differentiation
3.8 Adipogenic Differentiation
3.9 Cardiac Differentiation
3.10 Collagen Coating
3.11 Long-Term Storage of Cell Batches
4 Notes
References
Chapter 12: Role of Pericytes in the Development of the Renin/Angiotensin System: Induction of Functional Renin in Cultures of...
1 Introduction
2 Materials
2.1 Cell Isolation
2.1.1 Equipment
2.1.2 Materials
2.2 Primary Cell Culture
2.2.1 Equipment
2.2.2 Materials
2.3 Renin Studies
2.3.1 Equipment
2.3.2 Materials
Renin Induction in Cell Culture
Renin Activity Assay
3 Methods
3.1 Purification of Renal Pericytes Derived from Human Fetal Kidney
3.2 Kidney Pericyte Primary Culture and Their Basic Characterization
3.3 Surface Marker Analysis by Flow Cytometry
3.4 Differentiation of Renal Pericytes in Primary Culture into Renin-Producing Cells
3.4.1 Renin Induction Assay
3.4.2 Renin Activity Assay
4 Notes
References
Chapter 13: Metastatic Dissemination Mimicked in a Multicellular Transwell Assay
1 Introduction
2 Materials
2.1 Cells
2.2 Reagents
2.3 Supplies
2.4 Equipment
3 Method
3.1 Seeding of Endothelial Cells on Transwell Membrane (Day 1)
3.1.1 Coating of Transwell Membrane
3.1.2 Preparation of Endothelial Cell Suspension and Cell Culture on Transwell Membranes
3.2 Seeding of Pericytes on Transwell Membrane (Day 2, See Note 6)
3.3 Preparation of Tumor Cell Suspension and Immunofluorescent Staining
3.4 Fixation and Analysis of Transmigrated Tumor Cells
3.5 Alternative Variation of the Assay
4 Notes
References
Index