Human pluripotent stem cell derived organoid models /

Detalles Bibliográficos
Clasificación:Libro Electrónico
Otros Autores: Spence, Jason R. (Jason Robert), 1977- (Editor )
Formato: Electrónico eBook
Idioma:Inglés
Publicado: Cambridge, MA : Academic Press, 2020.
Colección:Methods in cell biology ; v. 159.
Temas:
Stem cells.
Stem Cells
Cellules souches.
Stem cells
Internet Resources.
Index not Present.
Acceso en línea:Texto completo
Tabla de Contenidos:
  • 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

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