Main description:

This detailed book encapsulates the most up-to-date methods of the intestinal stem cell field and provides guidance on a variety of techniques for studying intestinal stem cells properties. Beginning with a section on in vitro techniques to study different aspects of the intestinal stem cell functions by innovative imaging and functional assays, the volume continues with chapters detailing the single-cell transcriptional profiling method, the isolation of intestinal crypts to generate and establish 3D organoids, as well as different animal models of gastrointestinal cancer and examples of the use of in vivo methods for studying intestinal tumor-initiating cells or cancer stem cells. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and state-of-the-art, Intestinal Stem Cells: Methods and Protocols aims to provide comprehensive and easy to follow protocols designed to be helpful to both seasoned researchers and newcomers to this dynamic field.

Contents:

Part I: Characterization, Imaging, and Functional Assays

1. Identification and Isolation of Human LGR5+ Cells Using an Antibody-Based Strategy

Michael K. Dame, Sha Huang, Durga Attili, Jason R Spence, and Justin A. Colacino

2. Immune-Mediated Specific Depletion of Intestinal Stem Cells

Stephen E. Sherman and Judith Agudo

3. Analysis of Aged Dysfunctional Intestinal Stem Cells

Kodandaramireddy Nalapareddy and Hartmut Geiger

4. Strategies for Measuring Induction of Fatty Acid Oxidation in Intestinal Stem and Progenitor Cells

Chia-Wei Cheng, Omer H. Yilmaz, and Maria M. Mihaylova

5. Visualization of Stem Cell Niche by the Fluorescence Lifetime Imaging Microscopy

Irina A. Okkelman, Jens Puschhof, Dmitri B. Papkovsky, and Ruslan I. Dmitriev

6. Generation and Quantitative Imaging of Enteroid Monolayers

Laura E. Sanman, Ina W. Chen, Jake M. Bieber, Curtis A. Thorne, Lani F. Wu, and Steven J. Altschuler

7. Autophagy Detection in Intestinal Stem Cells

Jumpei Asano, Taku Sato, and Toshiaki Ohteki

Part II: Single-Cell Transcriptional Profiling of the Small Intestinal Epithelium

8. Single-Cell Transcriptional Profiling of the Intestinal Epithelium

Claudia Capdevila, Ruben I. Calderon, Erin C. Bush, Kismet Sheldon-Collins, Peter A. Sims, and Kelley S. Yan

9. Single-Cell Studies of Intestinal Stem Cell Heterogeneity during Homeostasis and Regeneration

Maxim Norkin, Claudia Capdevila, Ruben I. Calderon, Tianhong Su, Maria Trifas, Paloma Ordonez-Moran, and Kelley S. Yan

Part III: Organoids and Applications

10. Large-Scale Production of Recombinant Noggin and R-Spondin1 Proteins Required for the Maintenance of Stem Cells in Intestinal Organoid Cultures

David L. Hacker and Paloma Ordonez-Moran

11. Primary Intestinal Epithelial Organoid Culture

Tomohiro Mizutani and Hans Clevers

12. In Vivo Human PSC-Derived Intestinal Organoids to Study Stem Cell Maintenance

Simon Vales, Holly M. Poling, Nambirajan Sundaram, Michael A. Helmrath, and Maxime M. Mahe

13. Generation of Knock-Out Gene-Edited Human Intestinal Organoids

Chathruckan Rajendra, Tomas Wald, Kevin Barber, Jason R. Spence, Faranak Fattahi, and Ophir D. Klein

14. Direct Lineage Reprogramming of Mouse Fibroblasts to Acquire the Identity of Fetal Intestine-Derived Progenitor Cells

Shizuka Miura and Atsushi Suzuki

15. Single Molecule RNA FISH in Whole-Mount Organoids

Costanza Borrelli and Andreas E. Moor

16. Specific Gene Expression in Lgr5+ Stem Cells by Using Cre-Lox Recombination

Pierre Dessen, Joerg Huelsken, and Paloma Ordonez-Moran

17. Generating and Utilizing Murine Cas9-Expressing Intestinal Organoids for Large Scale Knockout Genetic Screening

Hossein Kashfi, Nicholas Jinks, and Abdolrahman Shams Nateri

Part IV: In Vivo Models

18. Mouse Model for Sporadic Mutation of Target Alleles to Understand Tumor Initiation and Progression and Stem Cell Dynamics

Theresa Nguyen, Elise Manalo, Taryn Kawashima, and Jared M. Fischer

19. Hemagglutinating Virus of Japan Envelop (HVJ-E)-Guided Gene Transfer to the Intestinal Epithelium

Masamichi Imajo

20. An Intrasplenic Injection Model for the Study of Cancer Stem Cells Seeding Capacity

Caroline Dafflon, Albert Santamaria-Martinez, and Paloma Ordonez-Moran

21. Organoid Derivation and Orthotopic Xenotransplantation for Studying Human Intestinal Stem Cell Dynamics

Shinya Sugimoto, Masayuki Fujii, and Toshiro Sato

22. Advanced Colorectal Cancer Orthotopic Patient-Derived Xenograft Models for Cancer and Stem Cell Research

Irene Chicote, Juan Antonio Camara, and Hector G. Palmer

23. Modeling Colorectal Cancer Progression through Orthotopic Implantation of Organoids

Felipe de Sousa e Melo, Jonathan M. Harnoss, Noelyn Kljavin, Ryan Scott, Catherine Sohn, Kevin G. Leong, and Frederic J. de Sauvage