Preface

Abstract

The concept of tissue regeneration was already present in ancient Greece, reflected by the mythological stories of Prometheus or the Hydra, and described by Aristotle. The first scientific studies of the phenomenon were performed around 1740 by Abraham Trembley on the cnidarian polyp Hydra. Yet, it took another 150 years until the idea emerged that tissue maintenance, turnover, and regeneration may be rooted in rare cells with unique properties: stem cells. During the past 50 years, the development and improvement of techniques to isolate, track, manipulate, culture, characterize, and transplant cells has led to the discovery of stem cells in many different tissues. The power of the hematopoietic stem cell to repopulate the entire blood system, first demonstrated by Till and McCulloch in 1963, has long since been harnessed for clinical use. More recently, the identification of the even more versatile pluripotent embryonic stem cell by Evans and Kaufman (1981) and Martin (1981) has revolutionized our ability to probe mammalian developmental biology and to model human diseases. In recent years the fascination of scientists with stem cells has spilled over into the public domain, and many share the hope that the 21st century will see a revolution in regenerative medicine as novel therapies are derived from stem cells. Continued scientific study of the biology of stem cells will be critical for this prospect to become a reality. It is the goal of the editors, in developing this manual, to facilitate this endeavor by providing scientists with a compendium of well established protocols in stem cell biology. Along with the continued progress of the field of stem cell biology, this collection of protocols will expand. The manual is written such that even a seasoned stem cell biologist will find many novel and useful ideas, but with enough detail provided to also guide those with less experience. This product is not intended to substitute for a graduate course in stem cell biology or for a comprehensive textbook in the field. Introductory texts on stem cells and cell and developmental biology that we recommend include Handbook of Stem Cells (Lanza et al., 2004), Developmental Biology (Gilbert, 2006), and Molecular Cell Biology (Lodish et al., 2004) or Molecular Biology of the Cell (Alberts et al., 2002). We also strongly recommend that readers gain first-hand experience in basic laboratory techniques and safety procedures by training in a well established laboratory. Finally, with the great promise and potential of stem cells, come ethical concerns. We urge stem cell biologists to reflect on these issues and to respect internationally accepted ethical guidelines and limitations such as those developed by the International Society for Stem Cell Research on the Conduct of Human Embryonic Stem Cell Research (ISSCR; see APPENDIX A.1A). Subjects in this manual are organized by chapters, which are subdivided into sections that contain protocols organized in units. Protocol units, which constitute the bulk of the title, generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method. Other units present more general information in the form of explanatory text with no protocols. Overview units contain theoretical discussions that lay the foundation for subsequent protocols, while discussion units present more general information. Page numbering reflects the modular arrangement by unit; for example, page 1A.2.3 refers to Chapter 1 (Embryonic and Extraembryonic Stem Cells), Section A (Isolation of Embryonic Stem Cells, UNIT Unavailable (Derivation of hESCs from Intact Blastocysts), and page 3 of that particular unit. Although many reagents and procedures are employed repeatedly throughout the manual, we have opted to retain individual authors' recipes or supplier designations because of the importance of using a particular reagent or procedure for successful stem cell experiments. Cross-referencing among the units is used for very basic procedures that do not vary from laboratory to laboratory. Because this publication is first and foremost a compilation of laboratory techniques in stem cell biology, we have included explanatory information where required to help readers gain an intuitive grasp of the procedures. Some sections begin with special overview units that describe the state of the art of the topic matter and provide a context for the procedures that follow. Section and unit introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters, troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key references to inform users of particularly useful background reading, original descriptions, or applications of a technique. Many units in the manual contain groups of protocols, each presented with a series of steps. One or more basic protocols are presented first in each unit and generally cover the recommended or most universally applicable approaches. Alternate protocols are provided where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the basic protocol. Support protocols describe additional steps that are required to perform the basic or alternate protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual or because they are performed in a time frame separate from the basic protocol steps. Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, while others are solutions unique to a particular protocol. Recipes for solutions are provided in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., RIPA buffer) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. Throughout the manual, the authors have recommended commercial suppliers of chemicals, biological materials, and equipment. It is recommended that the user follow the author's designations; often those are the products that the author, after considerable experimentation, has found will work under the particular conditions. In other cases, the experience of the author of that protocol is limited to that brand. In the latter situation, recommendations are offered as an aid to the novice in obtaining the tools of the trade. Anyone carrying out these protocols may encounter the following hazardous or potentially hazardous materials: (1) radioactive substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, and (3) pathogenic and infectious biological agents. Check the guidelines of your particular institution with regard to use and disposal of these hazardous materials. Although cautionary statements are included in the appropriate units, we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials must be used in strict accordance with local and national regulations. Many protocols call for use of live animals (usually rats or mice) for experiments. Prior to conducting any laboratory procedures with live subjects, the experimental approach must be submitted in writing to the appropriate Institutional Animal Care and Use Committee (IACUC) or must conform to appropriate governmental regulations regarding the care and use of laboratory animals. Written approval from the IACUC (or equivalent) committee is absolutely required prior to undertaking any live-animal studies. Some specific animal care and handling guidelines are provided in the protocols where live subjects are used, but check with your IACUC or governmental guidelines to obtain more extensive information. See the International Society for Stem Cell Research “Guidelines for the Conduct of Human Embryonic Stem Cell Research,” reproduced in APPENDIX A1.1. Research using human tissues must be reviewed and approved by the independent institutional ethics review panel, and donated material must be provided voluntarily with informed consent. Most of the protocols included in this manual are used routinely in the authors' laboratories. These protocols work for them; to make them work for you the authors have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers' observations and suggestions. We encourage readers to send their comments and questions to currentprotocols@wiley.com. Another valuable resource is the companion website http://www.currentprotocols.com. This site features tools, calculators, apps, troubleshooting tips, webinars, and videos to assist in the experiments presented in this manual. The individual article pages provide valuable summary information and easy access to the content. While the editorial board enlists contributors for most of the topics presented in this manual, we invite individuals to submit letters of intent describing new topics that they would like to publish in CPSC. The editorial board will carefully consider suggested topics and invite authors to submit full manuscripts in some cases. Letters of intent may be submitted by email to currentprotocols@wiley.com. This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the hard work by the Current Protocols editorial staff at John Wiley and Sons. We are extremely grateful for the critical contributions by Kathy Morgan (Developmental Editor), who kept the editors and the contributors on track and played a key role in bringing the entire project into existence. Other skilled members of the Current Protocols staff who contributed to the project include Joseph White, Tom Cannon, and Sheila Kaminsky. The extensive copyediting required to produce an accurate protocols manual was ably handled by Allen Ranz, Susan Lieberman, Marianne Huntley, and Sylvia de Hombre. Typesetting and electronic illustrations were prepared by Aptara.