Bookedited by Jason R. Spence.
Contents:
Intro
Human Pluripotent Stem Cell Derived Organoid Models
Copyright
Contents
Contributors
Chapter 1: Generation of esophageal organoids and organotypic raft cultures from human pluripotent stem cells
1. Introduction
2. Significance and applications
3. Morphological and transcriptional analysis of developing esophageal cultures
4. Overview of differentiation protocol
5. Step-by-step protocol
5.1. Coating plates for stem cell culture maintenance and directed differentiation
5.1.1. Materials and reagents
5.1.2. Protocol 5.2. Differentiation of stem cells into esophageal organoids
5.2.1. Materials, reagents and equipment
5.2.2. Solutions preparation
5.2.3. Protocol (Fig. 2A)
5.2.3.1. hPSC dissociation and plating in 24-well plate (day-1)
5.2.3.2. Differentiation protocol
5.2.3.3. Embedding foregut spheroids in Matrigel
5.2.3.4. Reduction of HEOs density
5.3. Esophageal raft culture protocol
5.3.1. Materials and reagents
5.3.2. Coating 100mm plates with collagen IV
5.3.3. Dissociation of HEOs and culturing in keratinocyte media 5.3.4. Preparing collagen-Mouse fibroblasts gels for raft cultures
5.3.5. Transferring cells from keratinocyte media to raft cultures
5.4. Immunofluorescence analysis of HEOs and organotypic raft cultures (Fig. 4)
5.4.1. Materials and reagents
5.4.2. Tissue fixation, preparation and cryosection
5.4.3. Immunofluorescent protocol
6. Future directions
7. Summary
Acknowledgments
References
Chapter 2: Generation and use of gastric organoids for the study of Helicobacter pylori pathogenesis
1. Introduction
2. Significance and applications of gastric organoids 3. Overview of the protocol
4. Step-by-step protocol
4.1. Generation of human-derived gastric organoids from normal and tumor tissues
4.1.1. Materials and reagents
4.1.2. Procedure: Generation of human-derived normal gastric organoids
4.1.3. Procedure: Generation of human-derived tumor gastric organoids
4.2. Generation of human-PBMC derived immune cells
4.2.1. Materials and reagents
4.2.2. Procedure: Isolation of PBMCs from whole blood using Ficoll-Paque density gradient medium
4.2.3. Procedure: Isolation of PBMCs from whole blood using Lymphoprep 4.2.4. Procedure: Freezing of PBMCs
4.2.5. Procedure: Culture of dendritic cells (DCs)
4.2.6. Procedure: Culture of cytotoxic T lymphocytes (CTLs)
4.2.7. Procedure: Culture of myeloid-derived suppressor cells (MDSCs)
4.3. Organoid/immune cell co-culture
4.3.1. Procedure: DCs and CTL co-culture
4.3.2. Procedure: Organoid-immune cell co-culture
4.4. Orthotopic transplantation
4.4.1. Materials and reagents
4.4.2. Procedure: Orthotopic transplantation of human-derived gastric organoids
5. Precursor techniques
6. Safety considerations and standards