Imaging and Tracking Stem Cells: Methods and Protocols

Preface
Contents
Contributors
Molecular Imaging and Tracking Stem Cells in Neurosciences
	1 Introduction
	2 Molecular Imaging Modalities Using Magnetic Resonance Imaging
	3 Molecular Imaging Modalities Using Positron Emission Tomography
	4 Molecular Imaging Modalities Using Bioluminescence Imaging
	5 Some Problems Associated with Positron Emission Tomography Molecular Imaging
	6 Conclusions
	References
Tracking Germline Stem Cell Dynamics In Vivo in C. elegans Using Photoconversion
	1 Introduction
	2 Materials
		2.1 Humid Chambers to Store Slides with 10% Agarose Pads
		2.2 Reagents for Making Slides with 10% Agarose Pads
		2.3 Reagents for Live Worm Mounting and Recovery
		2.4 Equipment for Photoconversion and Imaging
	3 Methods
		3.1 Preparation of the Humid Chamber for Storing Slides with 10% Agarose Pads (Fig. 2)
		3.2 Preparation of Slides with 10% Agarose Pads
		3.3 Mounting of Live Worms on 10% Agarose Pad Slides Using a Microbead Solution
		3.4 Imaging of Live Worms and Photoconversion
		3.5 Recovery of Worms from Slides After Imaging
		3.6 Remounting of Worm(s) for Subsequent Imaging
	4 Notes
	References
Long-Term Cell Fate Tracking of Individual Renal Cells Using Serial Intravital Microscopy
	1 Introduction
	2 Materials
		2.1 Material for AIW Preparation
		2.2 Surgical Instruments and Equipment
		2.3 Serial Intravital Imaging
		2.4 Ex Vivo Histology Preparation
	3 Methods
		3.1 Abdominal Imaging Window (AIW) Preparation
		3.2 Surgery Preparation
		3.3 Surgical AIW Implantation
		3.4 Serial Intravital Imaging
		3.5 Serial Intravital Imaging
		3.6 In Vivo Imaging Followed by Ex Vivo Histology of the Same Cortical Kidney Regions
		3.7 Reusing of the AIW
		3.8 Application of Serial Intravital MPM of the Mouse Kidney
		3.9 Summary
	4 Notes
	References
Tracking and Imaging of Transplanted Stem Cells in Animals
	1 Introduction
	2 Materials
		2.1 Stem Cells and Culture Condition
		2.2 Cell Labeling with Imaging Agents
		2.3 In Vivo Animal Imaging Systems
		2.4 Animal Models
	3 Methods
		3.1 Stem Cell Culture and Labeling
		3.2 In Vivo Imaging and Tracking of Transplanted Stem Cells to Injured Tissue and Diseases (Fig. 1)
		3.3 In Vivo Imaging of Stem Cell Recruitment for Tissue Engineering Applications
	4 Notes
	References
Generation and Analysis of Pluripotent Stem Cell-Derived Cardiomyocytes and Endothelial Cells for High Content Screening Purpo
	1 Introduction
	2 Materials
		2.1 Generation and Maintenance of Human-Induced Pluripotent Stem Cell (hiPSC)
		2.2 Generation and Maintenance of Human-Induced Pluripotent Stem Cell-Derived Endothelial Cells (hiPSC-ECs)
		2.3 Generation and Maintenance of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)
		2.4 Antibodies and Vital Dyes
		2.5 Apoptotic Agent
		2.6 Image Acquisition and Analysis
	3 Methods
		3.1 Human-Induced Pluripotent Stem Cell (hiPSCs)
			3.1.1 hiPSC Generation from Peripheral Blood Mononuclear Cells (Fig. 2)
			3.1.2 Passaging hiPSCs Cultured on Mouse Embryonic Fibroblast (MEF) Feeder Cells
			3.1.3 Freezing hiPSCs Cultured on MEF
			3.1.4 Culturing hiPSCs Growing in Monolayer
			3.1.5 Passaging hiPSC Monolayer Cultures (See Note 8)
				Accutase
				Versene
				ReLeSR
			3.1.6 Freezing hiPSC Monolayer Cultures
			3.1.7 Thawing hiPSCs
			3.1.8 Immunocytochemistry for Pluripotent Markers (Fig. 3)
		3.2 Human-Induced Pluripotent Stem Cell-Derived Endothelial Cells (hiPSC-ECs)
			3.2.1 Endothelial Cell Differentiation (Fig. 4)
			3.2.2 Endothelial Cell Sorting by Fluorescence-Activated Cell Sorting (FACS)
			3.2.3 Replating hiPSC-ECs
			3.2.4 Freezing hiPSC-ECs
			3.2.5 Thawing hiPSC-ECs
			3.2.6 Immunocytochemistry for Endothelial Markers (Fig. 5)
			3.2.7 Tube Formation Assay (Fig. 6)
			3.2.8 Endothelial Metabolic Assay (Fig. 7)
		3.3 Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)
			3.3.1 Cardiomyocyte Differentiation (Fig. 8)
			3.3.2 Replating hiPSC-CMs
			3.3.3 Freezing hiPSC-CMs
			3.3.4 Thawing hiPSC-CMs
			3.3.5 Immunocytochemistry for Cardiac Markers (Fig. 9)
			3.3.6 Live Cell Staining for Cell Death Assay for hiPSC-CMs (Fig. 10)
			3.3.7 Algorithm for High Content Imaging of Cell Death and Hypertrophy Assay on Live hiPSC-CMs (Based on [4, 6])
	4 Notes
	References
Covisualization of Global DNA Methylation/Hydroxymethylation and Protein Biomarkers for Ultrahigh-Definition Epigenetic Phenot
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Equipment
	3 Methods
		3.1 Pretreatment of Cells
		3.2 First Immunofluorescence
		3.3 Acid Treatment
		3.4 Second Immunofluorescence
		3.5 Counterstaining and Mounting
			3.5.1 For Super-Resolution Imaging
		3.6 Image Acquisition and Visualization
	4 Notes
	References
A Distinctive MRI-Based Absolute Bias Correction Protocol for the Potential Labelling and In Vivo Tracking of Stem Cells in a
	1 Introduction
		1.1 Relevant Processes Involved in This Protocol
			1.1.1 Isolation of MSCs from the Mouse Bone Marrow
			1.1.2 Induction of Traumatic Brain Injury in the Mouse Model
			1.1.3 Synthesis of Iron Oxide Contrast Agent
			1.1.4 MSC Labelling with the Contrast Agent
			1.1.5 Stem Cell Tracking by MRI
	2 Materials
		2.1 Mice
		2.2 Reagents
		2.3 Equipment
		2.4 Reagents Setup
		2.5 Equipment Setup
			2.5.1 Animal MRI System
			2.5.2 Traumatic Brain Injury Induction Setup
	3 Methods
		3.1 Isolation and Culture of MSCs from Mice Bone Marrow: Timing 3-4 Weeks
		3.2 Preparation of Magnetic Nanoparticles: Timing 4-5 Days
		3.3 Labelling of MSCs with Iron Oxide Contrast Agent: Timing 10-11 h
		3.4 Induction of Traumatic Brain Injury in Mice: Timing 16-20 h for 5 Mice
		3.5 Infusion of Labelled MSCs: Timing 10 min per Mouse
		3.6 Longitudinal Stem Cell Tracking Using 7T MRI Scanner: Timing Variable
		3.7 T2 Time Measurement in Injured and Normal Area of Brain: Timing Variable
		3.8 Advantages of the Protocol
		3.9 Limitations of the Protocol
	4 Notes
	References
Efficient Labeling of Human Mesenchymal Stem Cells Using Iron Oxide Nanoparticles
	1 Introduction
	2 Materials
		2.1 Fabrication of Mono-Dispersed Iron Oxide Nanoparticles (IONPs)
		2.2 Establishment of Stem-Cell Culture for Labeling
		2.3 Cell Fixation
		2.4 Prussian Blue (PB) Stain Preparation
	3 Methods
		3.1 Fabrication of Iron Oxide Nanoparticles
		3.2 Transformation of Hydrophobic IONPS to Hydrophilic for Biological Application
		3.3 Characterization of IONPs
		3.4 Labeling of hBM-MSCs with IONPs
		3.5 Prussian Blue (PB) Staining of IONPs-Labeled Cells
	4 Notes
	References
Imaging and Tracking Stem Cell Engraftment in Ischemic Hearts by Near-Infrared Fluorescent Protein (iRFP) Labeling
	1 Introduction
	2 Materials
		2.1 Lentivirus Packaging
		2.2 Stem Cell Labeling
		2.3 Myocardial Infarction Model and Intramyocardial Cell Transplantation
		2.4 Imaging and Tracking Stem Cells in Infarct Hearts
		2.5 Immunohistochemistry Identification of Engrafted Stem Cells in Ischemic Myocardium
	3 Methods
		3.1 Lentivirus Packaging
		3.2 Stem Cell Labeling
		3.3 Myocardial Infarction Model and Intramyocardial Cell Transplantation
		3.4 Imaging and Tracking Stem Cells in Infarct Hearts
		3.5 Identifying Engrafted Stem Cells in Ischemic Myocardium by Confocal Fluorescent Microscopy
	4 Notes
	References
Long-Term Intravital Imaging of the Cornea, Skin, and Hair Follicle by Multiphoton Microscope
	1 Introduction
	2 Materials
		2.1 Multiphoton Microscopic Imaging Platform
		2.2 Transgenic Mice Expressing Fluorescent Proteins in Specific Cell Populations
		2.3 Long-Term Life Support System
		2.4 Customized Stages for Live Imaging
	3 Methods
		3.1 Animal Preparation for In Vivo Imaging of the Cornea, Skin, and HFs
			3.1.1 Live Imaging of the Cornea
			3.1.2 Live Imaging of Epidermis and HFs
		3.2 Imaging Parameters and Processing
			3.2.1 Imaging Process for 3D Images
			3.2.2 Imaging Process for 4D Images
	4 Notes
	References
Cell Cycle Analysis Using In Vivo Staining of DNA-Synthesizing Cells
	1 Introduction
	2 Materials
		2.1 Buffers and Reagents
		2.2 Instruments
	3 Methods
		3.1 Double Sequential Labelling of DNA-Synthesizing Cells by EdU and BrdU In Vivo
			3.1.1 EdU and BrdU In Vivo Administration
			3.1.2 Immunophenotyping of Bone Marrow Cells and Detection of the Incorporated EdU and BrdU (See Note 4)
			3.1.3 Flow Cytometry Data Analysis
			3.1.4 Limitations
		3.2 Cell Flow Rate of DNA-Labelled Cells Arising from Mitosis
			3.2.1 BrdU In Vivo Administration
			3.2.2 Immunophenotyping of Bone Marrow Cells and Detection of BrdU-Labelled Cells with 2n DNA Content
			3.2.3 Flow Cytometry Data Analysis
			3.2.4 Limitations
	4 Notes
	References
Metabolic Labeling of Live Stem Cell for In Vitro Imaging and In Vivo Tracking
	1 Introduction
	2 Materials
		2.1 hUCB-EPCs Isolation and Culture
		2.2 Metabolic Labeling Agents
		2.3 Western Blot Analysis for Analysis of Metabolic Labeling Efficiency
		2.4 Stem Cell Imaging for Confocal and FACS
	3 Methods
		3.1 hUCB-EPCs Culture
		3.2 In Vitro Cell Labeling Using Metabolic Labeling Agents
		3.3 Western Blot Analysis for Analysis of Labeling Efficiency
		3.4 Stem Cell Imaging After Treatment of Metabolic Labeling Agents Using Confocal
		3.5 Stem Cell Imaging After Treatment of Metabolic Labeling Agents Using FACS
	4 Notes
	References
Study of Intracellular Cargo Trafficking and Co-localization in the Phagosome and Autophagy-Lysosomal Pathways of Retinal Pigm
	1 Introduction
	2 Materials
		2.1 Isolating POS from Porcine Eyes
		2.2 POS Feeding Assay
		2.3 Immunofluorescence Staining
	3 Methods
		3.1 Isolating POS from Porcine Eyes
		3.2 Tagging POS with FITC
		3.3 POS Feeding Assay
		3.4 Analysis Using Fiji and Volocity
		3.5 Visualization of Images Using Amira Software
	4 Notes
	References
Time-Lapse Video Microscopy and Single Cell Tracking to Study Neural Cell Behavior In Vitro
	1 Introduction
	2 Materials
		2.1 A Poly-d-Lysine Hydrobromide (PDL) Stock Solution and the Coating of Plates
		2.2 Culture Medium
		2.3 Post-imaging Immunocytochemistry
		2.4 Microscopy
		2.5 Tracking System
	3 Methods
		3.1 Cell Culture and Plating of the Selected Neural Population or Cell Lineage
		3.2 Live Imaging by Time-Lapse Video Microscopy
		3.3 Post-imaging Immunocytochemistry, Data Collection, and Processing
		3.4 Single Cell Tracking
		3.5 Final Outcome
	4 Notes
	References
Multiphoton Microscopy for Noninvasive and Label-Free Imaging of Human Skin and Oral Mucosa Equivalents
	1 Introduction
		1.1 Multiphoton Microscopy
			1.1.1 Two-Photon Excited Fluorescence (2PEF)
			1.1.2 Second Harmonic Generation (SHG)
	2 Materials
		2.1 Microscope for TPEF and SHG
			2.1.1 Laser
			2.1.2 Microscope and Its Ancillaries
		2.2 Cells
		2.3 Culture Components
	3 Methods
		3.1 Culture of Fibroblasts and Keratinocytes
		3.2 Fabrication of Full-Thickness Skin and Oral Mucosa Equivalents
		3.3 Microscope Setup
		3.4 2PEF Imaging of Keratinocytes and Fibroblasts
		3.5 SHG Imaging of Collagen
		3.6 Noninvasive Imaging of 3D Organotypic Cultures of Skin and Oral Mucosa
	4 Notes
	References
Molecular Imaging of Therapeutic Effect of Mesenchymal Stem Cell-Derived Exosomes for Hindlimb Ischemia Treatment
	1 Introduction
	2 Materials
		2.1 Mesenchymal Stem Cells Culture
		2.2 MSC-Exosomes Isolation
		2.3 Vegfr2-luc-KI Mice and Murine Hindlimb Ischemia Models
		2.4 MSC-Exosomes Treatment
		2.5 D-Luciferin Preparation
		2.6 Bioluminescence Imaging of Therapeutic Effect of Exosomes In Vivo
	3 Methods
		3.1 Mesenchymal Stem Cells Culture
		3.2 MSC-Exosomes Isolation
		3.3 Vegfr2-luc-KI Mice and Murine Hindlimb Ischemia Models
		3.4 MSC-Exosomes Treatment
		3.5 D-Luciferin Preparation
		3.6 Bioluminescence Imaging of Therapeutic Effect of MSC-Exosomes In Vivo
	4 Notes
	References
In Vitro Methods to Simulate Pollution and Photo-Pollution Exposure in Human Skin Epidermis
	1 Introduction
	2 Materials
		2.1 Adult Normal Human Epidermal Keratinocytes
			2.1.1 Determining DPE and CSC Doses for Treating Keratinocytes
			2.1.2 Active Exposure to Particulate Matter
			2.1.3 Passive Exposure to Particulate Matter
		2.2 Normal Human Keratinocytes Prepared from Neonate Foreskins
			2.2.1 PAH and UVA1: Photo-Pollution Exposure
		2.3 3D Skin Models
			2.3.1 Active Exposure to Particulate Matter
			2.3.2 Passive Exposure to Particulate Matter
			2.3.3 PAH and UVA1: Photo-Pollution Exposure
		2.4 Validation of DPE or CSC Exposure in 2D and 3D Models
	3 Methods
		3.1 Adult Normal Human Epidermal Keratinocytes
			3.1.1 Determining DPE and CSC Doses for Treating Keratinocytes
			3.1.2 Active Exposure to Particulate Matter
			3.1.3 Passive Exposure to Particulate Matter
		3.2 Normal Human Keratinocytes Prepared from Neonate Foreskin
		3.3 PAH and UVA1: Photo-Pollution Exposure
		3.4 3D Skin Models
			3.4.1 Active Exposure to Particulate Matter
			3.4.2 Passive Exposure to Particulate Matter
			3.4.3 PAH and UVA1: Photo-Pollution Exposure
		3.5 Validation of DPE or CSC Exposure in 2D and 3D Models
			3.5.1 2D Models (NHEK Cells)
			3.5.2 3D Models (EpiSkin RHE)
	4 Notes
	References
Correction to: Long-Term Cell Fate Tracking of Individual Renal Cells Using Serial Intravital Microscopy
Index