Intestinal Differentiated Cells, 1st ed. 2023 Methods and Protocols Methods in Molecular Biology Series, Vol. 2650

This volume details the most up-to-date methods of intestinal differentiation program. Chapters guide readers through techniques to characterize intestinal differentiated cell, RNAseq, single-cell RNAseq transcriptional profiling methods, isolation of intestinal crypts, and methods for studying intestinal differentiated cancer cells. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and methods, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

Authoritative and cutting-edge, Intestinal Differentiated Cells: Methods and Protocols aims to be comprehensive guide for researchers.

Part I: CHARACTERIZATION, IMAGING AND FUNCTIONAL ASSAYS

1        Differentiated epithelial cells of the gut

Andrea Bonilla and Paloma Ordóñez-Morán

2        Identification of differentiated intestinal epithelial cells using immunostaining and fluorescence microscopy

Jessica R. Digrazia, Melinda A. Engevik, and Amy C. Engevik

Gurveer Marva, Seyda Ünsal, Andrew V. Benest, David O. Bates, and Paloma Ordóñez- Morán

4        In vivo model for isolating epithelial cells of the anorectal transition zone

Louciné Mitoyan, Charlyne Gard, Sébastien Nin, Béatrice Loriod and Géraldine Guasch

5        In vitro and in vivo assays for testing retinoids effect on intestinal progenitors’ lineage commitments

Krishna R. Gajera, Kathryn L. Fair, Gordon Moran, Nicholas R.F. Hannan, Joerg Huelsken and Paloma Ordóñez-Morán

Part II: TRANSCRIPTIONAL PROFILING OF INTESTINAL CELLS

6        TORNADO-seq: a protocol for high-throughput targeted RNA-seq based drug screening in organoids

7        Epigenetic and transcriptional dynamics of Notch program in intestinal differentiation

Shahadat Rahman, Xi Lan, Christopher Terranova, Rayan El-Kholdi, Omer H. Yilmaz, and Chia-Wei Cheng

8        Defining anorectal transition zone heterogeneity using single-Cell RNA sequencing

Louciné Mitoyan, Charlyne Gard, Sébastien Nin, Béatrice Loriod and Géraldine Guasch

Part III: IN VITRO CULTURE AND APPLICATIONS

9        Directed differentiation of murine and human small intestinal organoids towards all mature lineages

Adriana Martinez-Silgado, Joep Beumer and Hans Clevers

10    Modeling Notch activity and lineage decisions using intestinal organoids

Yifan Qiu, Sabrina K. Phanor, Subin Pyo, and Chia-Wei Cheng

Masamichi Imajo, Akira Hirota, and Shinya Tanaka

12    Visualization of differentiated cells in 3D- and 2D-intestinal organoid cultures        

Hikaru Hanyu, Shinya Sugimoto, and Toshiro Sato

13    In-vitro culture and histological evaluation of 3D organotypic cultures

William Dalleywater, Francesca Wheat, Declan Sculthorpe, Georgina Hyland, and Mohammad Ilyas

14    Fluorescence intensity and fluorescence lifetime imaging (FLIM) microscopies of cell differentiation in the small intestinal organoids using cholera toxin

Irina A. Okkelman and Ruslan I. Dmitriev

15    In vitro morphogenesis and differentiation of human intestinal epithelium in a gut-on-a-chip

Woojung Shin and Hyun Jung Kim

16    Co-culturing human intestinal enteroid monolayers with innate immune cells

Janet F. Staab, Jose M. Lemme-Dumit, Rachel Latanich, Marcella F. Pasetti, and Nicholas C. Zachos

Part IV: DIFFERENTIATION IN COLON CANCER

17    Measuring cell differentiation in colorectal cancer

Isabel Puig, Irene Chicote and Héctor G. Pálmer

18    Intestinal cell differentiation and phenotype in 2D and 3D cell culture models

Magda Martinez-Espuga, Alvaro Mata and Paloma Ordóñez-Morán

19    Confocal laser scanning imaging of cell junctions in human colon cancer cells

Peixun Zhou and M. Angeles Juanes

Hannah M. Brown and M. Angeles Juanes

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts