Intestinal Differentiated Cells: Methods and Protocols

Preface
Contents
Contributors
Part I: Characterization, Imaging and Functional Assays
Chapter 1: Differentiated Epithelial Cells of the Gut
1 Secretory Lineage
1.1 Paneth Cells
1.2 Enteroendocrine Cells
1.3 Goblet Cells
1.4 Tuft Cells
2 Absorptive Lineage
2.1 Enterocytes
2.2 Microfold Cells
References
Chapter 2: Identification of Differentiated Intestinal Epithelial Cells Using Immunostaining and Fluorescence Microscopy
1 Introduction
2 Materials
2.1 Deparaffinization of Tissue Sections
2.2 Antigen Retrieval
2.3 Protein Block
2.4 Mouse-on-Mouse Block
2.5 Primary Antibodies (See Notes 2 and 3)
2.6 Wash Steps
2.7 Secondary Antibodies
2.8 Nuclei Counterstain
2.9 Wash Steps
2.10 Mounting Slides
2.11 Imaging and Processing
3 Methods
3.1 Immunostaining Paraffin-Embedded Sections (See Note 1)
3.2 Slide Deparaffinization
3.3 Rehydration
3.4 Heat-Mediated Epitope Retrieval Using a Pressure Cooker
3.5 Blocking Nonspecific Antigens
3.6 Mouse-on-Mouse Block
3.7 Primary Antibodies
3.8 Wash Slides
3.9 Secondary Antibodies
3.10 Hoechst Staining
3.11 Wash Slides
3.12 Mounting Tissue Sections
3.13 Imaging
3.14 Image Processing
4 Notes
References
Chapter 3: Novel Approach to Measure Transepithelial Electrical Resistance in Intestinal Cells
1 Introduction
2 Materials
2.1 Cells Preparation
2.2 ECIS Measurements
3 Methodology
3.1 Preparation of the Array (Use Class II Laminar Flow Hood)
3.2 Cell Seeding
3.3 ECIS Measurements
4 Notes
References
Chapter 4: In Vivo Model for Isolating Epithelial Cells of the Anorectal Transition Zone
1 Introduction
2 Materials
2.1 Mice
2.2 Tissue Anorectal Isolation and Anal Glands Removal
2.3 Sample Preparation for Flow Cytometry (FACS)
3 Methods
3.1 Anorectal Tissue Isolation and Anal Glands Removal
3.2 Sample Preparation for FACS
3.3 FACS Sorting
4 Notes
References
Chapter 5: In Vitro and in Vivo Assays for Testing Retinoids Effect on Intestinal Progenitors´ Lineage Commitments
1 Introduction
2 Materials
2.1 In Vitro Assays
2.1.1 2D Experiments
2.1.2 3D Experiments
2.2 In Vivo Assay
3 Methods
3.1 In Vitro Assays
3.1.1 2D Experiments
3.1.2 3D Experiments
3.2 In Vivo Assays
3.2.1 Preparation of Cells Before Surgery
3.2.2 Generation of Patient-Derived Xenografts
4 Notes
References
Part II: Transcriptional Profiling of Intestinal Cells
Chapter 6: TORNADO-seq: A Protocol for High-Throughput Targeted RNA-seq-Based Drug Screening in Organoids
1 Introduction
2 Materials
2.1 mRNA Isolation
2.2 RT Reaction
2.3 Library Amplification (First- and Second-Stage PCR)
3 Methods
3.1 Designing the Panel of Genes of Interest
3.2 Designing the Primers for Targeted RNA-Seq for a Selected Number of Genes
3.3 Targeted RNA-seq Library Construction
3.3.1 mRNA Isolation
3.3.2 RT Reaction
3.3.3 Library Amplification (First- and Second-Stage PCR)
3.4 RNA Sequencing and Analysis
4 Notes
References
Chapter 7: Epigenetic and Transcriptional Dynamics of Notch Program in Intestinal Differentiation
1 Introduction
2 Materials
2.1 ChIP-seq Analysis for H3K27ac Profiling
2.2 Single-Cell RNA-seq for Lateral Inhibition and Stem-to-Paneth Differentiation
3 Methods
3.1 ChIP-seq Analysis for H3K27ac Profiling
3.1.1 Processing Intestinal Crypts for Cross-Linking
3.1.2 Lysis, Sonication, and Antibody Preparation
3.1.3 Chromatin Immunoprecipitation
3.1.4 Washing and Reverse Cross-Linking
3.1.5 ChIP-Sequencing Library Preparation
3.1.6 ChIP-seq Data Processing
3.1.7 Identification of ChIP-seq Binding Sites
3.1.8 Assigning Binding Sites to Genes
3.2 Lineage Tracing and Single-Cell RNA-seq for Lateral Inhibition and Stem-to-Paneth Differentiation
3.2.1 Sample Preparation and Sequencing
3.2.2 Data Processing and Cell Clustering
3.2.3 GSEA Analysis for Lateral Inhibition
3.2.4 Differential Expression of Cell-Type Signature Genes: Stem-to-Paneth Transition
4 Notes
References
Chapter 8: Defining Anorectal Transition Zone Heterogeneity Using Single-Cell RNA Sequencing
1 Introduction
2 Materials
2.1 Cell Counting
2.2 Chromium Single-Cell RNA Sequencing (10x Genomics)
2.3 Quantification and Quality Control
2.4 Sequencing
2.5 Bioinformatic Analysis
3 Methods
3.1 Single-Cell RNA Sequencing
3.2 Cell Counting
3.3 Chromium Single-Cell RNA Sequencing
3.4 GEM Generation and Barcoding
3.5 Post-GEM-RT Cleanup and cDNA Amplification
3.6 3′ Gene Expression Library Construction
3.7 Sequencing
3.8 Bioinformatic Analysis
4 Notes
References
Part III: In Vitro Culture and Applications
Chapter 9: Directed Differentiation of Murine and Human Small Intestinal Organoids Toward All Mature Lineages
1 Introduction
2 Materials
2.1 Mouse Small Intestinal Organoid Maintenance and Differentiation
2.2 Human Small Intestinal Organoid Maintenance and Differentiation
2.3 Mouse and Human Media Variations
2.4 RNA Isolation and Quantitative Real-Time PCR Assay (qPCR)
2.5 Immunofluorescence Staining
3 Methods
3.1 Near-Normal Ratios of Intestinal Lineages in Human Organoids
3.1.1 IF Medium Protocol
3.2 Directed Differentiation of Murine and Human Intestinal Organoids
3.3 qPCR-Mediated Validation of Differentiation
3.4 Immuno\fluorescence-Mediated Validation of Differentiation
4 Notes
References
Chapter 10: Modeling Notch Activity and Lineage Decisions Using Intestinal Organoids
1 Introduction
2 Materials
2.1 Assess Notch Signaling Using Hes1-GFP Reporter and γ-Secretase Inhibitor Treatment
2.2 Genetic Engineering for Gene Deletion and NICD Overexpression
2.3 Histological Alcian Blue Staining
3 Methods
3.1 Assess Notch Activity Using Hes1-GFP Reporter and γ-Secretase Inhibitor Treatment
3.2 Genetic Engineering for Gene Deletion and NICD Overexpression
3.3 Histological Alcian Blue Staining (See Note 5)
4 Notes
References
Chapter 11: Generation of Fetal Intestinal Organoids and Their Maturation into Adult Intestinal Cells
1 Introduction
2 Materials
2.1 Dissection of Mouse Embryos and Isolation of Fetal Intestinal Cells
2.2 Establishment and Passaging of Fetal Intestinal Spheroids
2.3 Analysis of the Maturation Status of Cultured Fetal Intestinal Cells
3 Methods
3.1 Dissection of Mouse Embryos
3.2 Isolation and Culture of Fetal Intestinal Epithelial Cells
3.3 Passaging of Fetal Intestinal Spheroids
3.4 Analysis of In Vitro Maturation of Fetal Intestinal Cells into Adult Intestinal Cells
4 Notes
References
Chapter 12: Visualization of Differentiated Cells in 3D and 2D Intestinal Organoid Cultures
1 Introduction
2 Materials
2.1 Intestinal Organoid Culture (3D)
2.2 Intestinal Organoid Culture (2D)
2.3 Whole-Mount Staining (3D)
2.4 Whole-Mount Staining (2D)
3 Methods
3.1 Intestinal Organoid Culture (3D)
3.2 Passage of Intestinal Organoids (3D)
3.3 Intestinal Organoid Culture (2D)
3.4 Whole-Mount Staining (3D)
3.5 Whole-Mount Staining (2D)
4 Notes
References
Chapter 13: In Vitro Culture and Histological Evaluation of 3D Organotypic Cultures
1 Introduction
2 Materials
2.1 3D Organotypic Culture (Collagen Gels)
2.2 Processing and Embedding Tissues
2.3 Microtomy
2.4 Preparing Slides for Histological Techniques
2.5 H + E Staining
2.6 Picrosirius Red Staining
2.7 Immunohistochemistry
2.8 Slide Dehydration and Mounting
3 Methods
3.1 3D Organotypic Culture
3.2 Tissue Processing and Embedding
3.3 Microtomy
3.4 Preparing Slides for Histology
3.5 Histological Staining
3.6 Immunohistochemistry
3.7 Dehydration and Mounting
4 Notes
References
Chapter 14: Fluorescence Intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of Cell Differentiation in the Small ...
1 Introduction
2 Materials
2.1 Small Intestinal Organoids
2.2 Chemicals, Plasticware, and Equipment
2.3 Microscopy Imaging Supplies
2.4 Preparation of Fixed Organoid Samples
2.5 Data Acquisition and Analysis Software
3 Methods
3.1 Defrosting the Intestinal Organoid Culture
3.2 Passaging of Intestinal Organoid Culture and Seeding for Microscopy Imaging
3.3 Sample Staining and Imaging Acquisition
3.4 Processing of Microscopy Data
3.5 Anticipated Results
3.5.1 Multiparametric Imaging of CTX Conjugates with Live Cell Markers of Proliferation and Lgr5-GFP-Positive Small Intestinal...
3.5.2 Effect of Vitamin D3 Treatment on CTX-A555 Fluorescence Lifetime
4 Notes
References
Chapter 15: In Vitro Morphogenesis and Differentiation of Human Intestinal Epithelium in a Gut-on-a-Chip
1 Introduction
2 Materials
2.1 Culture of Human Intestinal Epithelium
2.2 Fabrication of a Gut-on-a-Chip
2.3 Culture of Human Intestinal Epithelium in a Gut-on-a-Chip
2.4 Morphological Assessment
3 Methods
3.1 Culture of Human Intestinal Epithelium: Caco-2 Cells
3.2 Culture of Human Intestinal Epithelium: Intestinal Organoids
3.3 Fabrication of a Gut-on-a-Chip
3.4 Induction of 3D Intestinal Morphogenesis in a Gut-on-a-Chip
3.5 Morphological Assessment of Differentiated Epithelium in a Gut-on-a-Chip
4 Notes
References
Chapter 16: Co-culturing Human Intestinal Enteroid Monolayers with Innate Immune Cells
1 Introduction
2 Generating Confluent Human Enteroid Monolayers on Transwells
2.1 Three-Dimensional Enteroid Cultures
2.1.1 Materials
2.2 Seeding Enteroid Fragments onto Transwell Inserts
2.2.1 Materials
2.2.2 Enteroid Monolayer Formation
2.2.3 Alternate Monolayer Protocol
3 Isolation of Immune Cells from Human Peripheral Blood
3.1 Human Monocytes
3.1.1 Materials
3.1.2 Protocol
3.2 Human PMN
3.2.1 Materials
3.2.2 Protocol
4 Co-culture Assembly
4.1 Materials
4.2 Assembly of Immune-Enteroid Co-cultures
5 Notes
References
Part IV: Differentiation in Colon Cancer
Chapter 17: Identifying Cell Differentiation in Colorectal Cancer
1 Introduction
2 Materials
2.1 Patient-Derived Xenografts
2.2 Equipment and Tools for Patient-Derived Xenografts Methodology
2.3 Immunostaining
2.4 Equipment and Tools for the Immunostaining Methodology
3 Methods
3.1 Patient-Derived Xenografts
3.1.1 Fresh Patient Tissue
3.1.2 Mice Tumor Tissue
3.1.3 Cell Preparation
3.1.4 Subcutaneous Injection Procedure
3.2 Immunostaining on Formalin-Fixed Paraffin-Embedded Sections of Intestinal Tissue
4 Notes
References
Chapter 18: Intestinal Cell Differentiation and Phenotype in 2D and 3D Cell Culture Models
1 Introduction
2 Materials
2.1 Caco-2 Cas-9 RFP Culture (2D)
2.2 Caco-2 Cas-9 RFP Culture (3D)
2.3 Total RNA Purification
2.4 cDNA Synthesis
2.5 cDNA Amplification by qPCR
3 Methods
3.1 Caco-2 Cas9-RFP Cell Culture
3.2 RNA Isolation and cDNA Synthesis
3.3 Quantitative Real-Time PCR (qPCR)
4 Notes
References
Chapter 19: Confocal Laser Scanning Imaging of Cell Junctions in Human Colon Cancer Cells
1 Introduction
2 Materials
2.1 Cell Growth and Differentiation of Caco-2 Cells
2.2 Immunofluorescence
2.3 Equipment and Others
3 Methods
3.1 Growth and Differentiation of Caco-2 Cells
3.2 Immunofluorescence
3.3 Imaging Using Confocal Laser Scanning Microscopy
3.3.1 Conventional Confocal Mode (Fig. 2)
3.3.2 Lightning Mode (Fig. 2)
4 Notes
References
Chapter 20: Automated Quantitative Analysis of Shape Features in Human Epithelial Monolayers and Spheroids Generated from Colo...
1 Introduction
2 Materials/Equipment
3 Methods
3.1 Quantitative Analysis of Cell Shape Features of HCT116 Colorectal Cancer Cells Forming 2D Monolayers
3.2 Quantitative Analysis of Individual Cell Shape Parameters Forming 3D HCT116 Spheroids
4 Notes
References
Index