Programmed Morphogenesis: Methods and Protocols

Preface
Contents
Contributors
Part I: Understanding Signaling Events and Patterns
Chapter 1: TASBE Image Analytics: A Processing Pipeline for Quantifying Cell Organization from Fluorescent Microscopy
1 Introduction
2 Materials
3 Methodology
3.1 Step-by-Step Procedure
3.1.1 Input Marshaling
3.1.2 Segmentation
3.1.3 Filtering and Clustering
3.1.4 Calculation of Frame Statistics
3.1.5 Tracking
3.2 Validation
3.3 Example Results
4 Notes
4.1 Software Setup
4.2 Parameter Configuration
4.3 Threshold Parameters
4.4 Cluster Parameters
4.5 Debugging Parameters
References
Chapter 2: Neighborhood Impact Factor to Study Cell-Fate Decision-Making in Cellular Communities
1 Introduction
2 Materials
3 Methods
3.1 Image Preparation
3.2 CellProfiler Processing Pipeline
3.3 Import SQLite Database and Produce NIF Data
3.4 Produce Spatial Plot of Cell Expression
3.5 Analysis of NIF Results
3.5.1 Total Expression Impact Factor
3.5.2 Local Density Impact Factor
3.5.3 Distance Adjusted Impact Factor
3.6 Example of NIF Processing and Output in Human Fetal Liver Organoid
4 Notes
References
Chapter 3: A Quantitative Lineage-Tracing Approach to Understand Morphogenesis in Gut
1 Introduction
2 Materials
3 Methods
3.1 Plug Detection and Tamoxifen Injection
3.2 Cesarean Section (Fig. 4) (See Note 4)
3.3 Tissue Collection (Fig. 4) and Staining (Fig. 5)
3.4 Clonal Dynamics Quantification (Fig. 6)
4 Notes
References
Part II: Programming Signaling Events and Patterns
Chapter 4: Reconstitution of Morphogen Signaling Gradients in Cultured Cells
1 Introduction
1.1 General Method
1.2 Strengths
1.3 Weaknesses
2 Materials
2.1 Parental Cell Lines
2.2 Cell Culture Materials
2.2.1 NIH3T3 Cell Culture Media
2.2.2 NIH3T3 Imaging Media
2.2.3 General Tissue-Culture Supplies
2.2.4 Transfection Kit and Chemicals
2.2.5 Fluorescent Dye Control
2.2.6 Materials for Establishing and Imaging Gradients
2.3 Microscopy
3 Methods
3.1 Morphogen-Producing and -Detecting Cell Lines
3.2 Radial Gradients
3.3 Linear Gradients
3.4 Time-lapse Imaging
3.5 Imaging Controls
3.6 Image Analysis
4 Notes
References
Chapter 5: Engineering Shape-Controlled Microtissues on Compliant Hydrogels with Tunable Rigidity and Extracellular Matrix Lig...
1 Introduction
2 Materials
2.1 Fabricating Silicon Wafer Masters
2.2 Fabricating PDMS Stamps
2.3 Activating Coverslips
2.4 Fabricating Polyacrylamide Hydrogels
2.5 Biotinylating Fibronectin or Laminin
2.6 Microcontact Printing Polyacrylamide Hydrogels
3 Methods
3.1 Fabricating Silicon Wafer Masters
3.2 Fabricating PDMS Stamps
3.3 Preparing Polyacrylamide Hydrogel Culture Substrates
3.3.1 Activating Coverslips
3.3.2 Fabricating Polyacrylamide Hydrogels
3.4 Microcontact Printing of Biotinylated ECM Proteins on Polyacrylamide Gels
3.4.1 Biotinylating Fibronectin or Laminin
3.4.2 Microcontact Printing
4 Notes
References
Chapter 6: Engineering Biophysical Cues for Controlled 3D Differentiation of Endoderm Derivatives
1 Introduction
2 Materials
2.1 General Reagents
2.2 Differentiation Media
2.3 Equipment
3 Methods
3.1 hPSC Preparation for Encapsulation
3.2 Encapsulation of hPSCs in Alginate Capsules
3.3 Modifying Biophysical Cues by Modification of Alginate Substrate Properties
3.3.1 Alginate Surface Roughness and Stiffness Characterization
3.3.2 Diffusivity of Alginate Capsule
3.4 hPSC Differentiation into Pancreatic Islet Endocrine Cells
3.5 Characterization of hPSC Aggregates
3.5.1 Immunostaining of hPSC Aggregates
Sectioning and Imaging
Whole-Mount Imaging
3.5.2 Flow Cytometry
3.5.3 RT-qPCR
3.6 Alginate Array Fabrication for Quantifying Effects of Combinatorial Perturbations
3.7 Imaging 3D Aggregates in the Array using LICOR
3.8 Statistical Modeling to Decouple the Effect of Multiparametric Perturbation
3.8.1 Evaluating Importance of Cation Concentration
3.8.2 Evaluating Combined Importance of Cation Concentration and Culture Configuration
4 Notes
References
Chapter 7: Rewiring Endogenous Bioelectric Circuits in the Xenopus laevis Embryo Model
1 Introduction
2 Materials
2.1 Guide RNA Design
2.2 CRISPR-Cas9 Microinjection
2.3 Frog Handling and Obtaining Fertilized Eggs
2.3.1 Inducing Ovulation
2.3.2 Manual Egg Collection
2.3.3 Isolating Testes
2.3.4 Preparing Embryos for Microinjection
2.3.5 Dejellying Embryos
2.3.6 Microinjection and Embryo Handling
2.3.7 Bioelectric Measurements and Reagents
3 Methods
3.1 Isolating Testes and Sperm
3.2 Obtaining Fertilized Eggs
3.2.1 Inducing Ovulation
3.2.2 Collecting the Eggs
3.2.3 In Vitro Fertilization
3.3 CRISPR Injections
3.3.1 Dejellying
3.3.2 Micro Injection
3.4 Measuring Relative Resting Membrane Potentials
4 Notes
References
Chapter 8: Engineering the Spatiotemporal Mosaic Self-Patterning of Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 CRISPR Interference gRNA Creation and Line Generation
2.3 Forced Aggregation
2.4 Live Imaging and Analysis
3 Methods
3.1 Designing and Testing Guide RNAs for CRISPRi
3.2 Creation of Mixed Aggregates
3.3 Plate down of Aggregates and Induction of CRISPRi
3.4 Time Lapse Microscopy
3.5 Image Analysis
4 Notes
References
Part III: Early Developmental Engineering
Chapter 9: Fate-Patterning of 2D Gastruloids and Ectodermal Colonies Using Micropatterned Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 2D Gastruloid and Ectoderm Patterns
2.1.2 Additional Reagents Required for Ectoderm Pattern
2.2 Micropatterning
2.3 Immunofluorescence and Imaging
3 Methods
3.1 Base Media Preparation
3.2 Coat Micropatterned Surface with Laminin and Seed Cells
3.2.1 Prepare Laminin Coated Micropattern Surface
96-Well CYTOO Micropattern Plate
CYTOO Micropattern Chip
3.2.2 Seed Cells
3.3 Wash Nonspecifically Bound Cells
3.4 Differentiate
3.4.1 Gastruloid Differentiation
3.4.2 Patterned Ectoderm Differentiation
3.5 Fix Cells, Stain for Immunofluorescence, and Image
3.5.1 Common to CYTOO 96-Well Plates and Chips
4 Notes
References
Chapter 10: Gastruloids: Embryonic Organoids from Mouse Embryonic Stem Cells to Study Patterning and Development in Early Mamm...
1 Introduction
1.1 Gastruloids and Other Embryonic Organoids
2 Materials
2.1 Cell Lines Tested with this Protocol
2.2 Routine Culture Medium
3 Methods
3.1 Routine Cell Culture
3.2 Preparation of N2B27
3.3 Preparing Cells for Gastruloid Plating
3.4 Gastruloid Generation and Culture
3.4.1 0 h After Aggregation (AA): Cell Plating
3.4.2 48 h After Aggregation: Addition of Secondary Medium with Chiron
3.4.3 72 h After Aggregation: Removal of Chiron Pulse and Medium Change
3.4.4 96 h After Aggregation: Medium Change
3.4.5 120 h After Aggregation: Medium Change and Continued Culture
3.5 Removing Gastruloids for Downstream Applications
4 Notes
References
Part IV: Organoids, Tissue Barriers, and Disease Models
Chapter 11: A Synergistic Engineering Approach to Build Human Brain Spheroids
1 Introduction
2 Materials
2.1 Array Platform
2.2 Cell Culture
2.3 Immunohistochemistry
2.4 Scanning Electron Microscopy
2.5 ELISA
3 Methods
3.1 Fabricating the Brain Spheroids Array
3.1.1 Master Fabrication
3.1.2 PDMS Mold Fabrication
3.1.3 Brain Spheroids Array Assembly
3.2 Generating the Genetically Engineered AD hNPCs
3.3 Maintaining ReN Cells and hiPSCs
3.4 Generating Brain Spheroids from Stem Cells
3.5 Application I: Imaging Brain Spheroids Morphology
3.6 Application II: Monitoring Pathophysiology
3.7 Application III: High-Throughput Drug Screening of AD Brain Spheroids with Automated Readouts
4 Notes
References
Chapter 12: Directed Differentiation of Human Pluripotent Stem Cells for the Generation of High-Order Kidney Organoids
1 Introduction
2 Materials
2.1 Culture and Passage of hPSCs
2.2 Plating of hPSCs for Differentiation
2.3 Differentiation of hPSCs Toward IM-Committed Cells and Generation of Kidney Organoids
2.4 Flow Cytometry Analysis of Differentiation Markers
2.5 Immunocytochemistry Analysis of Differentiation Markers
2.6 Primary Antibodies
2.7 Secondary Antibodies and Other Reagents
2.8 Other Materials
3 Methods
3.1 Culture and Passage of hPSCs
3.2 Plating of hPSCs for Differentiation
3.3 Differentiation of hPSCs Toward Posterior Primitive Streak (PPS) and Intermediate Mesoderm (IM)
3.4 Formation and Culture of Kidney Organoids
3.5 Flow Cytometry Analysis of Differentiation Markers
3.6 Immunocytochemistry Analysis of Differentiation Markers
4 Notes
References
Chapter 13: Methods for Controlled Induction of Singular Rosette Cytoarchitecture Within Human Pluripotent Stem Cell-Derived N...
1 Introduction
2 Materials
2.1 Micropatterned Culture Substrate Fabrication
2.2 Cell Culture
2.3 Immunostaining
3 Methods
3.1 Generating Micropatterned Substrates
3.2 Human PSCs Culture
3.2.1 Matrigel Coating of TCPS Plates
3.2.2 hPSC Culture
3.3 Derivation of Hindbrain and Spinal Neuromesodermal (NMP) Progenitors
3.4 Generation of Micropatterned Forebrain NSC Tissues
3.5 Generation of Micropatterned Hindbrain and Spinal NCS Tissues
3.6 Immunostaining
4 Notes
References
Chapter 14: 3D Self-Organized Human Blood-Brain Barrier in a Microfluidic Chip
1 Introduction
2 Materials
2.1 Microfluidic Technology
2.2 Fibrin Gel
2.3 Cell Culture
2.4 Coating Solution and Growth Factor-Enriched Culture Media
2.5 Fluorescent Dextran Solution
3 Methods
3.1 Preparing and Filling Fibrin Gel with Cells
3.2 Hydrating and Coating Media Channels
3.3 Changing Medium
3.4 Seeding Endothelial Cells in Media Channels
3.5 Perfusing Microvasculature with Fluorescent Dextran
3.6 Quantification of Microvascular Permeability
3.7 Quantification of Microvascular Geometry
4 Notes
References
Chapter 15: Modeling the Complexity of the Metastatic Niche Ex Vivo
1 Introduction
1.1 Overview of Legacy LiverChip Microphysiological System Technology
1.2 Strengths and Limitations for Investigating Metastatic Disease
1.3 Applications
2 Materials
2.1 Legacy LiverChip Microphysiological System Setup and Preparation
2.2 Immunofluorescence
3 Methods
3.1 Legacy LiverChip Microphysiological System Preparation
3.1.1 Legacy LiverChip Plate Assembly
3.1.2 Priming
3.1.3 Coat Scaffolds
3.1.4 Legacy LiverChip Plate Construction
3.2 Creating Hepatic Tissue (Day 0)
3.3 Media Change (Day 1)
3.4 Metastatic Progression (Days 3-15)
3.5 Immunofluorescence Staining and Imaging
3.5.1 Harvesting and Fixing Scaffolds
3.5.2 Quantification of Cancer Burden
3.5.3 Detection of Proliferating Cancer Cells
3.6 Effluent Collection, Assays and Analyses
4 Notes
References
Chapter 16: Fabrication Method of a High-Density Co-Culture Tumor-Stroma Platform to Study Cancer Progression
Abbreviations
1 Introduction
2 Materials
2.1 Photolithography
2.2 Soft Lithography
2.3 Surface Treatment
2.4 3D Coculture of Tumor-Stroma Sample
2.5 Instruments
2.6 Software
3 Methods
3.1 Photolithography
3.2 Soft Lithography
3.3 Surface Treatment of the PDMS Holders
3.4 Surface Treatment of PDMS Stamps
3.5 Development of the 3D Tumor-Stroma Coculture Model
3.6 Microscopy for Subsequent Studies on Tumor Cell Behavior (e.g., Migration)
4 Notes
References
Part V: In Vivo Therapeutic Applications
Chapter 17: A Method for Organoid Transplantation and Whole-Mount Visualization of Post-Engraftment Vascularization
1 Introduction
1.1 Methodological Strengths and Weaknesses
1.2 Applications
2 Materials
2.1 Vasculogenic Hydrogel Preparation
2.2 Transplantation Materials
2.3 Lectin Perfusion and Whole-Mount Imaging Materials
3 Methods
3.1 Vasculogenic Hydrogel Preparation (Fig. 2)
3.2 Organoid Transplantation (Fig. 3)
3.3 Lectin Injection for Vascular Labeling (Fig. 5)
3.4 Whole-Mount Imaging (Fig. 6)
4 Notes
References
Chapter 18: High-Throughput Production of Platelet-Like Particles
1 Introduction
2 Materials
2.1 Cell Culture for MEG-01 Cells (ATCC CRL-2021)
2.2 Flow Cytometry
2.3 Imaging
2.4 Making Platelet-Like Particles
3 Methods
3.1 Cell Culture
3.2 Making Platelet-Like Particles
3.3 Flow Cytometry
3.3.1 Whole Cells
3.3.2 Platelet-Like Particles
3.4 Imaging MEG-01 Cells from Plates
4 Notes
References
Index