Stem Cell Tools and Other Experimental Protocols (eBook)

Irina Klimanskaya, Robert Lanza (Herausgeber)

eBook Download: EPUB

2006 | 1. Auflage
520 Seiten
Elsevier Science (Verlag)
978-0-08-046964-5 (ISBN)

Infos

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.

* 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

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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 "e;cookbook and handy illustrations. The collection of protocols includes the isolation and maintenance of stem cells from various species using "e;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. - 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

Inhalt

Front Cover 1
Stem Cell Tools and Other Experimental Protocols 4
Copyright Page 5
Dedication 6
Table of Contents 8
Contributors to Volume 420 10
Preface 12
Foreword 14
Volumes in Series 16
Section I: In Vitro Experimentation and Research Tools 40
Chapter 1: Human Embryo Culture 42
Introduction 42
Human Embryo Development 43
Human Embryo Culture: General Considerations 47
Protocols 48
Results 53
Acknowledgments 54
References 54
Chapter 2: Characterization and Evaluation of Human Embryonic Stem Cells 57
Introduction 57
Characterization of Undifferentiated hESCs 58
Conclusion 71
Acknowledgments 72
References 72
Chapter 3: Feeder-Free Culture of Human Embryonic Stem Cells 76
Introduction 77
Methods for Feeder-Free Culture of hESCs 83
Acknowledgments 86
References 86
Chapter 4: Transgene Expression and RNA Interference in Embryonic Stem Cells 88
Retrovirus Expression Vectors and Embryonic Stem Cells 89
RNA Interference and ESCs 90
siRNA Expression Vector Design 91
Retrovirus Production 92
Retroviral and Lentiviral Gene Transfer into Mouse and Human ESCs 93
Transgene and siRNA Expression in Mouse and Human ESCs 96
Biotechnological and Medical Applications 99
Acknowledgments 100
References 100
Chapter 5: Lentiviral Vector-Mediated Gene Delivery into Human Embryonic Stem Cells 103
Introduction 104
Development of Lentiviral-Based Vectors 104
Design of HIV-1-Based Vectors 105
Transduction of hESCs by Lentiviral-Based Vectors 106
Potential Applications of Gene Delivery into hESCs by Lentiviral-Based Vectors 108
Design of HIV-1-Based Vectors for Transduction of hESCs 108
References 119
Chapter 6: The Use of Retroviral Vectors for Gene Transfer into Hematopoietic Stem Cells 121
Introduction 121
Viral Pseudotype 121
Vector Design 122
Packaging Cell Lines 123
Clinical Applications 124
Generation of Retrovirus Packaging Cell Lines 125
Summary and Future Directions 133
Acknowledgments 135
References 135
Chapter 7: Engineering Embryonic Stem Cells with Recombinase Systems 139
Introduction 140
Site-Specific Recombination 140
Large Serine Recombinases 144
Designing Substrates for Site-Specific Recombination 144
Generation of Conditional Alleles 152
Recombinase-Mediated Cassette Exchange (RMCE) 158
Molecular Switches 159
Protocols 162
References 170
Chapter 8: Gene Trapping in Embryonic Stem Cells 175
Introduction 176
Common Types of Gene Trap Vectors in IGTC 177
A User's Guide to the International Gene Trap Consortium Resource 183
Investigator-Initiated Screens 187
References 198
Chapter 9: GeneChips in Stem Cell Research 201
Introduction 202
Protocol 203
ESC Differentiation 214
ESC Genes and In Vivo Regeneration 223
Cell Screening 228
Integration and Meta-Analysis of GeneChip and Microarray Data 230
Applications to Cancer 233
Conclusion 238
Summary 239
Acknowledgments 259
References 259
Further Reading 263
Chapter 10: Microarray Analysis of Stem Cells and Differentiation 264
Introduction 264
Overview of Microarray Technology 264
Total RNA Isolation and Clean-Up 269
RNA Amplification 270
Direct Labeling of RNA for Microarray Hybridization 272
Indirect Labeling of RNA for Microarray Hybridization 273
Microarray Hybridization 274
Array-Based Comparative Genomic Hybridization (Array CGH) 275
Data Analysis 276
Confirmation Studies 285
Examples of Microarray Experiments for Stem Cell Biology and Differentiation 286
Identification of Stemness 286
Differentiation 287
Stem Cell Niches 288
Future Directions 290
References 290
Chapter 11: Purification of Hematopoietic Stem Cells Using the Side Population 294
Introduction 294
Protocol of HSC Sorting with Hoechst 33342 Staining (SP) 298
References 302
Chapter 12: Cellular Reprogramming 304
Introduction 304
Streptolysin O-M ediated Cell Permeabilization and Uptake of Reprogramming Cell Extracts 309
Cell Permeabilization and the Reprogramming Reaction 310
Preparation of Reprogramming Cell Extracts 317
Acknowledgments 319
References 319
Section II: Tissue Engineering and Regenerative Medicine 324
Chapter 13: Tissue Engineering Using Adult Stem Cells 326
Introduction 326
Biomaterials 329
Angiogenic Factors 331
Adult Stem Cells for Tissue Engineering 332
Conclusion 338
References 338
Chapter 14: Tissue Engineering Using Human Embryonic Stem Cells 342
Introduction 342
Special Considerations When Using hESCs as the Cell Source for TE 343
Growing hESCs in Defined Animal-Free Conditions 343
Obtaining the Desired Cell Population 344
Choosing the Right Scaffold 345
Scaling Up a Regulatable Bioprocess 345
hESC-Derived Connective Tissue Progenitors for TE 346
Preparation of MEF Feeder Layers 347
Starting hESC Culture 347
Passaging hESCs 348
Choosing the Right Scaffold for Connective Tissue Engineering 350
Harvesting Samples for Analyses 351
Electron Microscopy 352
Histological Analysis 352
References 354
Chapter 15: Engineering Cardiac Tissue from Embryonic Stem Cells 355
Introduction 355
Liquid Collagen-Based Cardiac Tissue Engineering 360
Conclusions 376
References 376
Chapter 16: Mesenchymal Stem Cells and Tissue Engineering 378
MSCs: Definition and Therapeutic Promise 378
Isolation and Expansion of MSCs 380
Multilineage Differentiation of MSCS 384
Clinical Translation of MSC-Based Therapies 392
Conclusions 394
Acknowledgments 394
References 395
Chapter 17: Bone Reconstruction with Bone Marrow Stromal Cells 401
Introduction 401
Cell Source for Bone Engineering 402
Animal Models for BMSC-Mediated Bone Engineering 403
Clinical Application of Engineered Bone 410
Protocol for Dog Cranial Bone Engineering 412
Protocol for Goat Femoral Bone Engineering 414
Acknowledgment 417
References 418
Chapter 18: Engineering Three-Dimensional Tissue Structures Using Stem Cells 420
Introduction 420
Protocols for Differentiating ESCs 422
Protocol for Scaffold Seeding 423
References 428
Chapter 19: Immunogenicity of Embryonic Stem Cells and Their Progeny 430
Introduction 431
Generation of Trimera Mice 432
Transplantation of hESCs and Derivatives into Trimera Mice 436
Assessing the Immune Response against Engrafted hESCs 440
Results and Discussion 443
Acknowledgments 446
References 446
Chapter 20: Manufacturing Considerations for Clinical Uses of Therapies Derived from Stem Cells 449
Introduction 449
U S. Regulatory Framework for Therapies Derived from Stem Cells 450
The Need for a Science-Based Approach for the Product and the Process 451
Ancillary Materials 455
Residual Ancillary Materials in Final Product 457
Genetic Modification 457
Combination Products 458
Developing a Manufacturing Process That Is Consistent and Scalable 458
Aseptic Processing 459
Characterization 460
In-Process and Release Testing 460
Shipment 464
Handling of Cellular Product at Clinical Site 464
The Need for a Quality Program 465
Summary 467
References 468
Author Index 470
Subject Index 508

Textauszug

[1]

Human Embryo Culture

Amparo Mercader; Diana Valbuena; Carlos Simón

Abstract

Human embryonic stem cells (hESCs) are derived from preimplantation embryos. Approximately 60% of human embryos are blocked during in vitro development. Although statistics are inconclusive, experience demonstrates that hESCs are more effectively derived from high‐quality embryos. In this way, optimal human embryo culture conditions are a crucial aspect in any derivation laboratory. Embryos can be cultured solely with sequential media or cocultured on a monolayer of a given cell type.

This chapter explores general aspects of human embryonic development, the concept of sequential culture and coculture, and specific protocols and procedures in which the authors are experienced, including the results obtained.

Introduction

Human embryonic stem cells (hESCs) are derived from preimplantation‐stage embryos, a process that involves culturing embryos to the blastocyst stage (Thomson et al., 1998). HESCs have also been isolated from morula‐stage embryos (Strelchenko et al., 2004) and even from later‐stage blastocysts (7–8 days) (Stojkovic et al., 2004). Although hESC lines have been derived from embryos of poor quality (Mitalipova et al., 2003), it is clear that hESCs are more efficiently derived from high‐quality embryos (Oh et al., 2005; Simon et al., 2005).

To optimize embryo development in vitro, it is essential to adopt a global approach to the embryo culture system that takes into account the media, gas phase, type of medium overlay, culture vessel, incubation chamber, ambient air quality, and the embryologists themselves. The concept of an embryo culture system highlights the interactions that exist not only between the embryo and its physical surroundings but also with all the parameters present in the laboratory. Only by taking such a holistic approach we can optimize embryo development in vitro as the previous step for optimal hESC derivation.

Initially, the zygote cleaves into two daughter cells, which subsequently divide to form the morula 4 days later. The transcription of the embryonic genome first occurs between the four‐and eight‐cell stages (Braude et al., 1988), which constitutes a critical moment. Compaction of the individual blastomeres follows, and finally develop to the blastocyst stage. Approximately 40–50% of embryos arrest during in vitro development.

Today, human embryology laboratories are faced not only with a multitude of embryo culture media from which to choose but also with various possibilities of how to use defined media or coculture systems.

Human Embryo Development

In the laboratory, embryo development from oocyte retrieval to the blastocyst stage occurs as follows:

Day 0: The human oocyte is retrieved from the follicle.

Day 1: Fertilization day. Polar bodies and pronuclei are visualized. Only fertilized eggs with two polar bodies and two pronuclei are considered to be correctly fertilized.

Day 2: First cleavage. The embryos generally have two to four cells. Embryos are evaluated for number of blastomeres (n), rate (%) and type (n) of fragmentation, symmetry (n), compaction (n), and multi- nucleation and are classified accordingly (Fig. 1A). Example: an embryo with four cells, 10% of fragmentation equally distributed throughout, with blastomeres of a similar size, without compaction and with one cell with two nuclei is classified as 4, 10, III, 2, 0, 1 × 2.

Fig. 1 Embryo development. (A) Two‐cell embryo. (B) Eight‐cell embryo. (C) Compacted morula. (D) Expanded blastocyst.

Day 3: The embryos have six to eight cells and are evaluated as indicated previously (Fig. 1B).

Day 4: Subsequent divisions form a 16–32‐cell embryo: the morula stage. Individual blastomeres become indistinguishable as they come into close contact with each other. This phenomenon is named compaction. On day 4, the type of morula is classified as either morula or compacted morula (Fig. 1C).

Day 5: Spaces appear between the compacting cells, resulting in the formation of an external layer of cells, known as the trophoblast, and a group of centrally located cells, known as the inner cell mass (ICM). At this stage of development, the embryo is called a blastocyst (Fig. 1D).

Day 6: The blastocoelic cavity enlarges and causes the embryo to grow and begin to hatch out from the zona pellucida (ZP). Blastocyst expansion thins the ZP because of a series of expansions and contractions.

Blastocysts are classified morphologically as follows:

• Early blastocyst: when spaces appear between the compaction (Fig. 2A).

Fig. 2 Types of blastocyst. (A) Early. (B) Cavitated. (C) Expanded. (D) Hatched.

• Cavitated blastocyst: when the blastocoelic cavity is more than 50% of the total volume (Fig. 2B).

• Expanded blastocyst: when the blastocoelic cavity enlarges in size, the embryo and a monolayer, also known as the trophectoderm (TE), and an ICM can be differentiated (Fig. 2C).

• Hatching blastocyst: the embryo begins to hatch out of the ZP.

• Hatched blastocyst: the embryo is outside the zona pellucida (Fig. 2D).

The different parts of the blastocysts—ICM and TE—can also be classified morphologically.

Inner Cell Mass

There are four types of ICM:

A. Dense and compact with many cells (Fig. 3A).

Fig. 3 (A–D) Types of inner cell mass. (E–H) Types of trophectoderm.

B. Several cells and not compact (Fig. 3B).

C. Very few cells (Fig. 3C).

D. Absence of a true ICM (pseudoblastocyst) (Fig. 3D).

Trophectoderm

There are four types:

A. Complete, with a monolayer of cells; forming a cohesive epithelium (Fig. 3E).

B. Incomplete, with a lineal zone (Fig. 3F).

C. With few large cells (Fig. 3G).

D. With degenerated cells (Fig. 3H).

Example: An expanded blastocyst with an ICM of very few cells and a trophectoderm with a lineal zone is classified as BE (C, B).

Embryo development does not always follow an “ideal” pattern, sometimes becoming delayed or blocked because of low quality or accelerating inappropriately because of chromosomal abnormalities. Furthermore, morphology can vary considerably and is difficult to interpret at the expected developmental stage.

Human Embryo Culture: General Considerations

The dramatic changes in embryo physiology, nutrient requirements, and nutrient gradients in the female reproductive tract have led to the formulation of two culture media that are applied at different stages of human embryo development. This is the practice of sequential culture media. On the other hand, the concept of “cells helping cells,” extended throughout many areas of cell biology, has prompted embryologists to coculture human embryos in the presence of other types of cells (feeder cells), resulting in the development of the coculture system (Simon et al., 1999; Mercader et al., 2003).

Sequential Culture

Several detailed treatises have been written on the composition of embryo culture media, focusing particularly on four components: glucose, amino acids, ethylenediaminetetraacetic acid (EDTA), and macromolecules. Studies in mammals, including humans (Conaghan et al., 1993; Quinn, 1995), have demonstrated the importance of relatively high concentrations of pyruvate and lactate and a relatively low level of glucose in the early stages, whereas the opposite metabolic conditions have been shown to be required at the blastocyst stage. Amino acids contained in culture media enhance human embryo development up until the blastocyst stage (Devreker et al., 1998). In particular, a switch occurs in amino acid requirements during embryo development (Lane and Gardner, 1997). The beneficial effects of divalent cation EDTA in embryo culture media have been extensively reported (Mehta et al., 1990), although said benefits are confined to the cleavage stage (Gardner and Lane, 1996; Gardner et al., 2000a). A commonly used protein source in human IVF and embryo culture has been patient serum, which is added to the culture medium at a concentration of 5–20%. However, recombinant human serum albumin (HSA) is now available, eliminating the problems associated with transfusion and permitting the standardization of media formulation (Gardner et al., 2000b).

In general, the sequential culture is composed of two different media designed to meet metabolic requirements throughout embryo development. The first of these media is...

Produkt-Details

• Erscheint lt. Verlag: 12.12.2006
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie ► Pflege
• Themenwelt: Medizin / Pharmazie ► Physiotherapie / Ergotherapie ► Orthopädie
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Technik ► Medizintechnik
• ISBN-10: 0-08-046964-7 / 0080469647
• ISBN-13: 978-0-08-046964-5 / 9780080469645