Stem Cell Tools and Other Experimental Protocols - 1st Edition - ISBN: 9780123736512, 9780080469645

Stem Cell Tools and Other Experimental Protocols, Volume 420

1st Edition

Serial Volume Editors: Robert Lanza Irina Klimanskaya
Hardcover ISBN: 9780123736512
eBook ISBN: 9780080469645
Imprint: Academic Press
Published Date: 12th December 2006
Page Count: 520
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Table of Contents

  • Dedication
  • Contributors to Volume 420
  • Preface
  • Foreword
  • Volumes in Series
  • Section I: In Vitro Experimentation and Research Tools
    • [1]: Human Embryo Culture
      • Abstract
      • Introduction
      • Human Embryo Development
      • Human Embryo Culture: General Considerations
      • Protocols
      • Results
      • Acknowledgments
    • [2]: Characterization and Evaluation of Human Embryonic Stem Cells
      • Abstract
      • Introduction
      • Characterization of Undifferentiated hESCs
      • Conclusion
      • Acknowledgments
    • [3]: Feeder-Free Culture of Human Embryonic Stem Cells
      • Abstract
      • Introduction
      • Methods for Feeder-Free Culture of hESCs
      • Acknowledgments
    • [4]: Transgene Expression and RNA Interference in Embryonic Stem Cells
      • Abstract
      • Retrovirus Expression Vectors and Embryonic Stem Cells
      • RNA Interference and ESCs
      • siRNA Expression Vector Design
      • Retrovirus Production
      • Retroviral and Lentiviral Gene Transfer into Mouse and Human ESCs
      • Transgene and siRNA Expression in Mouse and Human ESCs
      • Biotechnological and Medical Applications
      • Acknowledgments
    • [5]: Lentiviral Vector–Mediated Gene Delivery into Human Embryonic Stem Cells
      • Abstract
      • Introduction
      • Development of Lentiviral-Based Vectors
      • Design of HIV-1–Based Vectors
      • Transduction of hESCs by Lentiviral-Based Vectors
      • Potential Applications of Gene Delivery into hESCs by Lentiviral-Based Vectors
      • Design of HIV-1–Based Vectors for Transduction of hESCs
    • [6]: The Use of Retroviral Vectors for Gene Transfer into Hematopoietic Stem Cells
      • Publisher Summary
      • Introduction
      • Viral Pseudotype
      • Vector Design
      • Packaging Cell Lines
      • Clinical Applications
      • Generation of Retrovirus Packaging Cell Lines
      • Summary and Future Directions
      • Acknowledgments
    • [7]: Engineering Embryonic Stem Cells with Recombinase Systems
      • Abstract
      • Introduction
      • Site-Specific Recombination
      • Large Serine Recombinases
      • Designing Substrates for Site-Specific Recombination
      • Generation of Conditional Alleles
      • Recombinase-Mediated Cassette Exchange (RMCE)
      • Molecular Switches
      • Protocols
    • [8]: Gene Trapping in Embryonic Stem Cells
      • Abstract
      • Introduction
      • Common Types of Gene Trap Vectors in IGTC
      • A User’s Guide to the International Gene Trap Consortium Resource
      • Investigator-Initiated Screens
    • [9]: GeneChips in Stem Cell Research
      • Abstract
      • Introduction
      • Protocol
      • ESC Differentiation
      • ESC Genes and In Vivo Regeneration
      • Cell Screening
      • Integration and Meta-Analysis of GeneChip and Microarray Data
      • Applications to Cancer
      • Conclusion
      • Summary
      • Acknowledgments
      • Appendix 1 Sample Analysis
    • [10]: Microarray Analysis of Stem Cells and Differentiation
      • Abstract
      • Introduction
      • Overview of Microarray Technology
      • Total RNA Isolation and Clean-Up
      • RNA Amplification
      • Direct Labeling of RNA for Microarray Hybridization
      • Indirect Labeling of RNA for Microarray Hybridization
      • Microarray Hybridization
      • Array-Based Comparative Genomic Hybridization (Array CGH)
      • Data Analysis
      • Confirmation Studies
      • Examples of Microarray Experiments for Stem Cell Biology and Differentiation
      • Identification of Stemness
      • Differentiation
      • Stem Cell Niches
      • Future Directions
    • [11]: Purification of Hematopoietic Stem Cells Using the Side Population
      • Abstract
      • Introduction
      • Protocol of HSC Sorting with Hoechst 33342 Staining (SP)
    • [12]: Cellular Reprogramming
      • Abstract
      • Introduction
      • Streptolysin O–Mediated Cell Permeabilization and Uptake of Reprogramming Cell Extracts
      • Cell Permeabilization and the Reprogramming Reaction
      • Preparation of Reprogramming Cell Extracts
      • Acknowledgments
  • Section II. Tissue Engineering and Regenerative Medicine
    • [13]: Tissue Engineering Using Adult Stem Cells
      • Abstract
      • Introduction
      • Biomaterials
      • Angiogenic Factors
      • Adult Stem Cells for Tissue Engineering
      • Conclusion
    • [14]: Tissue Engineering Using Human Embryonic Stem Cells
      • Abstract
      • Introduction
      • Special Considerations When Using hESCs as the Cell Source for TE
      • Growing hESCs in Defined Animal-Free Conditions
      • Obtaining the Desired Cell Population
      • Choosing the Right Scaffold
      • Scaling Up a Regulatable Bioprocess
      • hESC-Derived Connective Tissue Progenitors for TE
      • Preparation of MEF Feeder Layers
      • Starting hESC Culture
      • Passaging hESCs
      • Choosing the Right Scaffold for Connective Tissue Engineering
      • Harvesting Samples for Analyses
      • Electron Microscopy
      • Histological Analysis
    • [15]: Engineering Cardiac Tissue from Embryonic Stem Cells
      • Abstract
      • Introduction
      • Liquid Collagen–Based Cardiac Tissue Engineering
      • Conclusions
    • [16]: Mesenchymal Stem Cells and Tissue Engineering
      • Abstract
      • MSCs: Definition and Therapeutic Promise
      • Isolation and Expansion of MSCs
      • Multilineage Differentiation of MSCS
      • Clinical Translation of MSC-Based Therapies
      • Conclusions
      • Acknowledgments
    • [17]: Bone Reconstruction with Bone Marrow Stromal Cells
      • Abstract
      • Introduction
      • Cell Source for Bone Engineering
      • Animal Models for BMSC-Mediated Bone Engineering
      • Clinical Application of Engineered Bone
      • Protocol for Dog Cranial Bone Engineering
      • Protocol for Goat Femoral Bone Engineering (Zhu et al., 2006)
      • Acknowledgment
    • [18]: Engineering Three-Dimensional Tissue Structures Using Stem Cells
      • Abstract
      • Introduction
      • Protocols for Differentiating ESCs
      • Protocol for Scaffold Seeding
    • [19]: Immunogenicity of Embryonic Stem Cells and Their Progeny
      • Abstract
      • Introduction
      • Generation of Trimera Mice
      • Transplantation of hESCs and Derivatives into Trimera Mice
      • Assessing the Immune Response against Engrafted hESCs
      • Results and Discussion
      • Acknowledgments
    • [20]: Manufacturing Considerations for Clinical Uses of Therapies Derived from Stem Cells
      • Abstract
      • Introduction
      • U.S. Regulatory Framework for Therapies Derived from Stem Cells
      • The Need for a Science-Based Approach for the Product and the Process
      • Ancillary Materials
      • Residual Ancillary Materials in Final Product
      • Genetic Modification
      • Combination Products
      • Developing a Manufacturing Process That Is Consistent and Scalable
      • Aseptic Processing
      • Characterization
      • In-Process and Release Testing
      • Shipment
      • Handling of Cellular Product at Clinical Site
      • The Need for a Quality Program
      • Summary
  • Author Index
  • Subject Index


This is the third of three planned volumes in the Methods in Enzymology series on the topic of stem cells. This volume is a unique anthology of stem cell techniques written by experts from the top laboratories in the world. The contributors not only have hands-on experience in the field but often have developed the original approaches that they share with great attention to detail. The chapters provide a brief review of each field followed by a “cookbook” and handy illustrations. The collection of protocols includes the isolation and maintenance of stem cells from various species using “conventional” and novel methods, such as derivation of ES cells from single blastomeres, differentiation of stem cells into specific tissue types, isolation and maintenance of somatic stem cells, stem cell-specific techniques and approaches to tissue engineering using stem cell derivatives. The reader will find that some of the topics are covered by more than one group of authors and complement each other. Comprehensive step-by-step protocols and informative illustrations can be easily followed by even the least experienced researchers in the field, and allow the setup and troubleshooting of these state-of-the-art technologies in other laboratories.

Key Features

  • Provides complete coverage spanning from derivation/isolation of stem cells, and including differentiation protocols, characterization and maintenance of derivatives and tissue engineering
  • Presents the latest most innovative technologies
  • Addresses therapeutic relevance including FDA compliance and tissue engineering


Biochemists, cell and molecular biologists, and bioengineers working with stem cells.


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© Academic Press 2006
Academic Press
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About the Serial Volume Editors

Robert Lanza Serial Volume Editor

Robert Lanza, M.D. is currently Chief Scientific Officer at Advanced Cell Technology, and Adjunct Professor of Surgical Sciences at Wake Forest University School of Medicine. He has several hundred scientific publications and patents, and over 30 books, including Principles of Tissue Engineering (1st through 4th Editions), Methods of Tissue Engineering, Principles of Cloning (1st and 2nd Editions), Essentials of Stem Cell Biology (1st and 2nd Editions), XENO, Yearbook of Cell & Tissue Transplantation, One World: The Health & Survival of the Human Species in the 21st Century (as editor, with forewords by C. Everett Koop and former President Jimmy Carter), and Medical Science & the Advancement of World Health. Dr. Lanza received his B.A. and M.D. degrees from the University of Pennsylvania, where he was both a University Scholar and Benjamin Franklin Scholar. He is a former Fulbright Scholar, and studied as a student in the laboratory of Richard Hynes (MIT), Jonas Salk (The Salk Institute), and Nobel laureates Gerald Edelman (Rockefeller University) and Rodney Porter (Oxford University). He also worked closely (and coauthored a series of papers) with the late Harvard psychologist B.F. Skinner and heart transplant pioneer Christiaan Barnard. Dr. Lanza's current area of research focuses on the use of stem cells in regenerative medicine.

Affiliations and Expertise

Advanced Cell Technology, MA, USA

Irina Klimanskaya Serial Volume Editor

Affiliations and Expertise

Advance Cell Technology, Worcester, MA, USA