Methods in Cancer Stem Cell Biology

✍ Scribed by Said M. Afify, Masaharu Seno

• Publisher: Springer
• Year: 2023
• Tongue: English
• Leaves: 251
• Edition: 1st ed. 2023
• Category: Library

✦ Synopsis

This book describes the use of stem cells and cancer stem cell generation in the inflammatory microenvironment (cancer-inducing niche) using induced pluripotent stem cells. It provides step-by-step techniques and manuals for studying stem cell and cancer stem cell generation with different applications in cancer research.  

The development of induced pluripotent stem cells has provided a new approach to studying cancer initiation by producing cancer stem cells without introducing mutations or foreign genes. The book features the research of the authors’ group, which was the first to generate cancer stem cells from stem cells in the presence of inflammatory conditions.  

The 20 chapters of this book cover topics such as generating pluripotent stem cells, converting normal stem cells to cancer stem cells, enriching, isolating and evaluating cancer stem cells. Methods for evaluating the characteristics of cancer stem cells and possible therapies against them are also discussed. 

The book provides easy-to-follow protocols that help researchers in the study of cancer stem cells. Illustrations help readers understand how the method of cancer stem cell generation can be applied as an essential method for assessing the carcinogenic potential of various non-mutagenic compounds. It will be a useful resource for graduate students, researchers, technicians, and physicians working in academic, hospital, and pharmaceutical settings. 

✦ Table of Contents

Preface
Acknowledgments
Contents
Chapter 1: On the Origin of Cancer
1.1 Background
1.2 Humoral Theory
1.3 Lymph Theory
1.4 Blastema Theory
1.5 Chronic Irritation Theory
1.6 Viral Carcinogenesis
1.7 Mutation Theory
1.8 Tissue Organization Field Theory (TOFT)
1.9 Stem Cells
1.10 Cancer Stem Cell Theory
1.11 Origin of the Cancer Stem Cell
1.11.1 Cell Fusion
1.11.2 Mutation
1.11.3 Epigenetics
1.11.3.1 Non-stem Cancer Cells
1.11.3.2 Stem Cells
References
Chapter 2: Culture of Cells: Basic Principles
2.1 Introduction
2.2 Basic Requirements of Cells in Culture
2.2.1 Equipments
2.2.1.1 Laminar Flow Hood
2.2.1.2 Cell Culture Incubators
2.2.1.3 Cleaning and Sterilization Facilities
2.2.2 Reagent Essential for Cell Culture
2.2.2.1 Media
2.2.2.2 Serum
2.2.3 Basic Techniques of Cell Culture
2.2.3.1 Cell Culture
2.2.3.2 Reviving the Cell
2.2.3.3 Passage
2.2.3.4 Preparation of frozen cell storage
References
Chapter 3: Stem Cell Culture from Embryos
3.1 Introduction
3.2 Materials
3.2.1 Equipment
3.2.2 Equipment
3.2.3 Reagent Preparation
3.3 Methods
3.3.1 Preparation of Gelatin-Coated Dishes
3.3.2 Preparation of MEF-Coated Dishes
3.3.3 Obtaining Blastocyst Stage Embryos
3.3.4 Thawing and Plating ESCs
3.3.5 Splitting ESCs on MEF
3.3.6 Passage ESC on Feeder-Less Dish
3.4 Notes
References
Chapter 4: Reprogramming of Normal Cells into Human Pluripotent Stem Cells
4.1 Introduction
4.2 Materials
4.2.1 Reagents
4.2.2 Equipment
4.2.3 Reagent Preparation
4.3 Methods
4.3.1 Preparation of Healthy Donor and Patient peripheral blood mononuclear cells (PBMC) Samples
4.3.1.1 Ficoll Density Gradient Separation Method
4.3.1.2 Cell Preparation Tubes
4.3.2 Preparation of Gelatin-Coated Dishes
4.3.3 Preparation of MEF-Coated Dishes
4.3.4 Generation of iPS Cells
References
Chapter 5: Maintenance of Human Pluripotent Stem Cells
5.1 Introduction
5.2 Materials
5.2.1 Reagents
5.2.2 Equipment
5.2.3 Reagent Preparation
5.3 Methods
5.3.1 Preparation of Gelatin-Coated Dishes
5.3.2 Preparation of MEF-Coated Dishes
5.3.3 Reviving Human iPSCs
5.3.4 Passage of Human iPSCs on MEF
5.3.5 Passage of Human iPSCs on Matrigel
5.3.6 Preparation of Cryopreservation Medium for iPSCs
References
Chapter 6: Identification of Cancer Stem Cells by Different Molecular Markers
6.1 Introduction
6.2 Materials
6.2.1 Reagents
6.2.2 Equipment
6.2.3 Reagent Preparation
6.3 Methods
6.3.1 Preparation of Primary Cells from a Tumor Tissue
6.3.2 Preparation of Cells from a Cancer Cell Line
6.3.3 Passage of Cancer Cells
6.3.4 Identification of CSCs by Cell Surface Markers
6.3.5 Separation of CSC Subpopulation
References
Chapter 7: Enrichment of Cancer Stem Cell from Malignant Tumor
7.1 Introduction
7.2 Materials
7.2.1 Reagents
7.2.2 Equipment
7.2.3 Reagent Preparation
7.3 Methods
7.3.1 Enrichment CD44 Expressing Subpopulation
7.3.1.1 Culture-Adherent Culture in Presence of Hyaluronic Acid (HA)
7.3.1.2 Suspension Culture
7.3.1.3 Passaging Spheres
7.3.2 Cell Preparation for Injection
References
Chapter 8: Isolation of Single Clonal Cell from Primary Cultured Cells and Establishment of a Cancer Stem Cell Line
8.1 Introduction
8.2 Materials
8.2.1 Reagents
8.2.2 Equipment
8.2.3 Reagent Preparation
8.3 Methods
8.3.1 Primary Culture of the Malignant Tumor Derived from U-251MG Spheroids (Continued from Chap. 7)
8.3.2 CSC Isolation
8.3.2.1 Fluorescence-Activated Cell Sorting (FACS)
8.3.2.2 Single Cell Cloning
8.3.3 Evaluation of Self-Renewal Potential of the Isolated Cells
8.3.4 Evaluation of Tumorigenic Potential of the Isolated Cells
References
Chapter 9: Artificial Generation of Cancer Stem Cells from Human Stem Cells
9.1 Introduction
9.2 Materials
9.2.1 Reagents
9.2.2 Equipment
9.2.3 Reagent Preparation
9.3 Methods
9.3.1 Preparation of Gelatin-Coated Dishes
9.3.2 Preparation of MEF-Coated Dishes
9.3.3 Reviving Human iPSCs
9.3.4 Passage of Human iPSCs on MEF
9.3.5 Passage of Human iPSCs on Matrigel
9.3.6 Preparation of Conditioned Medium (CM) for Conversion
9.3.7 Conversion of Human iPSCs on Matrigel
References
Chapter 10: Quick Method to Assess Non-mutagenic Carcinogens with iPS Cells
10.1 Introduction
10.2 Materials
10.2.1 Reagents
10.2.2 Equipment
10.2.3 Reagent Preparation
10.3 Methods
10.3.1 Conditioned Medium (CM) Preparation
10.3.2 Preparation of Dishes with Feeder Cells
10.3.3 Plating Mouse iPSCs on Feeder Cells
10.3.4 Transfer Mouse iPSCs to Feeder-Less Culture
10.3.5 Evaluation of Carcinogenicity in Vitro
10.3.6 Treating CSCs with the Compounds
10.3.7 Confirmation of Tumorigenicity by Transplantation into Nude Mice
References
Chapter 11: Self-renewal Potential of Cancer Stem Cells
11.1 Introduction
11.2 Materials
11.2.1 Reagents
11.2.2 Equipment
11.2.3 Reagent Preparation
11.3 Methods
11.3.1 Preparation of Gelatin-Coated Dishes
11.3.2 Reviving CSC
11.3.3 Sphere Formation Protocol
11.3.4 Immunofluorescence Staining
11.3.5 Passage Spheres
11.3.6 Hanging Drop Sphere Formation
11.3.7 Extreme Limiting Dilution Assay (ELDA)
References
Chapter 12: Differentiation Potential of Cancer Stem Cells In Vitro
12.1 Introduction
12.2 Materials
12.2.1 Reagents
12.2.2 Equipment
12.2.3 Reagent Preparation
12.2.3.1 Stem Cell Medium
12.2.3.2 Tube Medium
12.3 Methods
12.3.1 Preparation of Gelatin-Coated Dishes
12.3.2 Reviving CSC
12.3.3 Endothelial Differentiation
12.3.3.1 Tube Formation Assay
12.3.3.2 Immunostaining against CD31
12.3.4 Adipogenic Differentiation and Nile Red Staining
References
Chapter 13: Tumor Angiogenesis by Cancer Stem Cells In Vivo
13.1 Introduction
13.2 Materials
13.2.1 Reagents
13.2.2 Equipment
13.2.3 Reagent Preparation
13.2.3.1 Stem Cell Medium
13.3 Methods
13.3.1 Reviving CSC
13.3.2 Incubation of the Fertilized Eggs (EDD-0)
13.3.3 Puncture the Egg and Albumin Removal
13.3.4 Opening a Window in the Egg over CAM
13.3.5 Inoculation of CSCs on CAM
13.3.6 Observation of Tumor Angiogenesis
References
Chapter 14: Invasion and Metastatic Potential of Cancer Stem Cells In Vitro
14.1 Introduction
14.2 Materials
14.2.1 Reagents
14.2.2 Equipment
14.2.3 Reagent Preparation
14.2.3.1 Medium for Cancer Stem Cells Culture
14.2.3.2 Matrigel Preparation
14.2.3.3 PBS (pH 7.40) Preparation
14.2.3.4 0.1%(W/V) Gelatin
14.3 Methods
14.3.1 Preparation of Gelatin-Coated Dishes
14.3.2 Cancer Stem Cell Thawing
14.3.3 Cancer Stem Cell Passage
14.3.4 Cell Migration
14.4 Cell Invasion
References
Chapter 15: Metastatic Potential of Cancer Stem Cells In Vivo
15.1 Introduction
15.2 Materials
15.2.1 Reagents
15.2.2 Equipment
15.2.3 Reagent Preparation
15.2.3.1 Medium for Cancer Stem Cells Culture
15.2.3.2 PBS (pH 7.40) Preparation
15.2.3.3 0.1%(W/V) Gelatin
15.3 Methods
15.3.1 Preparation of Gelatin-Coated Dishes
15.3.2 Preparation of Cancer Stem Cells
15.3.3 Intra-Splenic Transplantation of Cancer Stem Cells
15.3.4 Detection of Hepatic Metastasis
15.3.5 Intravenous Injection of Tumor Cells
15.3.6 Detection of Lung Metastasis
References
Chapter 16: Anchorage-Independent Cell Growth Assay for Cancer Stem Cells: Tumorigenic Assay in Vitro
16.1 Introduction
16.2 Materials
16.2.1 Reagents
16.2.2 Equipment
16.2.3 Reagent Preparation
16.3 Methods
16.3.1 Preparation of Gelatin-Coated Dishes
16.3.2 Cancer Stem Cell Thawing
16.3.3 Preparation of Base Agar Layer
16.3.4 Cancer Stem Cell Preparation
16.3.5 Plating the Upper Layer of Agar Containing Cells
References
Chapter 17: Tumorigenic Potential of Cancer Stem Cells In Vivo
17.1 Introduction
17.2 Materials
17.2.1 Reagents
17.2.2 Equipment
17.2.3 Reagent Preparation
17.3 Methods
17.3.1 Preparation of Gelatin-Coated Dishes
17.3.2 Cancer Stem Cell Thawing
17.3.3 Preparation and Injection of miPS-LLCcm Cells
17.3.4 Tumor Fixation, Paraffin Embedding, and Section Preparation
17.3.5 Histochemical Observation with Hematoxylin and Eosin (H&E) Staining
17.3.6 Immunohistochemistry for the Malignant Tumor
17.4 Primary Culture from Malignant Tumor
References
Chapter 18: Development of Immunoliposomes Using Monoclonal Antibodies Targeting Cancer Stem Cells
18.1 Introduction
18.2 Materials
18.3 Methods
18.3.1 Preparation of Anti-human CD44 mab
18.3.2 Preparation of Liposome Encapsulating gPTX (gPTX-L)
18.3.2.1 Preparation of gPTX-L
18.3.2.2 Preparation of Immunoliposomes Containing gPTX (gPTX-IL)
18.3.2.3 Evaluation of Antitumor Effects of Drugs In Vivo
References
Chapter 19: In Vitro Evaluation of Anti-Cancer Stem Cell Drugs
19.1 Introduction
19.2 Materials
19.2.1 Reagent
19.2.2 Equipment
19.2.3 Reagent Preparation
19.2.3.1 MTT Solution
19.2.3.2 Phosphate-Buffered Saline Solution (PBS)
19.2.3.3 Trypan Blue Solution
19.3 Methods
19.3.1 Cancer Stem Cells Culture
19.3.2 Cell Plating for MTT
19.3.3 Drug Preparation
19.3.4 Formazan Formation
19.3.5 Data Analysis
References
Chapter 20: In Vitro Tumoroid Model Using Cancer Stem Cells
20.1 Introduction
20.2 Materials
20.2.1 Reagents
20.2.2 Equipment
20.2.3 Reagents setup
20.2.3.1 N-Acetyl-L-Cysteine
20.2.3.2 Nicotinamide
20.2.3.3 ROCK Inhibitor
20.2.3.4 Trypsin Solution
20.2.3.5 Medium for Cancer Stem Cells Culture
20.2.3.6 Matrigel Preparation
20.2.3.7 PBS (pH 7.40) Preparation
20.2.3.8 0.1%(w/v) Gelatin
20.3 Methods
20.3.1 Reviving Cancer Stem Cells
20.3.2 Sphere Formation
20.3.3 Cancer Organoid Development
20.3.4 Passage of the Organoids
20.3.5 Freezing of Cancer Organoids
References