Methods in Molecular Biology 1235 Ivan N. Rich Editor Stem Cell Protocols M ETHODS IN MOLECULAR BIOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Stem Cell Protocols Edited by Ivan N. Rich HemoGenix, Inc., Colorado Springs, CO, USA Editor Ivan N. Rich HemoGenix, Inc. Colorado Springs , CO , USA ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1784-6 ISBN 978-1-4939-1785-3 (eBook) DOI 10.1007/978-1-4939-1785-3 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014954236 © Springer Science+Business Media New York 2015 This work is subject to copyright. 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Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Pref ace Primary stem cells represent a very small and unique population of cells. Indeed, the proportion of stem cells compared to other cells in an organ or tissue is so small that they are usually morphologically unidentifi able. Yet, their presence can be detected by functional properties and characteristics that make stem cells unique. The present volume in this suc- cessful series of Methods in Molecular Biology on Stem Cell Protocols focuses on methods that allow primary stem cells from a variety of sources to be isolated, cultured in vitro, detected, and measured for specifi c applications. These applications range from those in basic, stem cell and veterinary research to toxicology and cellular therapy and regenerative medicine. If the volume demonstrates a slight bias towards the blood-forming system, it is no coincidence. More is known about the blood-forming or hematopoietic system than prob- ably any other primary stem cell system. The hematopoietic system has been used as a model for many stem cell systems. However, although every stem cell system is unique in its own way, there are many commonalities that are demonstrated in the methods and tech- niques described in this volume. All of these unique properties and characteristics are discussed and examined, mostly at the cellular level and in detail in this volume. Unlike non-defi nitive stem cell systems such as embryonic stem (ES) and induced pluripotent stem (iPS) cells, primary defi nitive stem cells still represent a black box of unknown biology and physiology. Although ES and iPS cells can be manipulated to produce large numbers of stem cells and functionally mature cells, such as hepatocytes, cardiomyocytes, and neurons, investigators still have to demon- strate that these artifi cially produced parent and progeny cells are similar in virtually every aspect to their primary counterparts. It is therefore imperative to understand and characterize primary stem cell populations and how they are regulated. Primary, defi nitive stem cells emit and receive a multitude of signals, both internal and external, to maintain homeostasis. For applications such as cel- lular therapy and regenerative medicine, it is important to understand the complexity of the stem cell compartment in order to develop assays that provide informative and predictive information prior to use in the patient. Assays are already available for this purpose. But, new future technologies, such as nanotechnology, may provide the capability, accuracy, and sensitivity needed to probe far deeper into the intricacies of the stem cell compartment. The question is whether these new technologies will be accepted and trusted over other tech- nologies that may have been in place for decades. v vi Preface Those new to the fi eld of stem cell biology will fi nd a wealth of “go to” and “how to” information that will hopefully provide some of the fundamentals to spark further interest and incentive to develop new technologies that will be required for basic and clinical research in the future. Those who have worked and are established in the fi eld will hopefully fi nd some new methodologies that will provide a wider viewpoint and an even greater scope for their own research. The editor wishes to thank all the authors who contributed their time and effort to this volume and stem cell biology fi eld. Colorado Springs, CO Ivan N. Rich Contents Preface. v Contributors . ix 1 Short Primer in Stem Cell Biology . 1 Ivan N. Rich 2 Measurement of Hematopoietic Stem Cell Proliferation, Self-Renewal, and Expansion Potential . 7 Ivan N. Rich 3 Measuring the Aging Process in Stem Cells . 19 Yi Liu, Gary Van Zant, and Ying Liang 4 Measuring the Potency of a Stem Cell Therapeutic . 33 Holli Harper and Ivan N. Rich 5 Culturing Protocols for Human Multipotent Adult Stem Cells . 49 Bart Vaes, Sara Walbers, Kristel Gijbels, David Craeye, Robert Deans, Jef Pinxteren, and Wouter van’t Hof 6 Isolation of Murine Bone Marrow Scavenging Sinusoidal Endothelial Cells . 59 Peter A. G. McCourt, Ana Oteiza, Benjamin Cao, and Susan K. Nilsson 7 Analysis of Circadian Rhythms in Embryonic Stem Cells . 73 Jiffin K. Paulose, Edmund B. Rucker III, and Vincent M. Cassone 8 Measuring Stem Cell Circadian Rhythm . 81 William Hrushesky and Ivan N. Rich 9 Cryopreservation of Human Pluripotent Stem Cells: A General Protocol. 97 Takamichi Miyazaki and Hirofumi Suemori 10 Biological Differences Between Native and Cultured Mesenchymal Stem Cells: Implications for Therapies . 105 Elena Jones and Richard Schäfer 11 The Use of Multiparameter Flow Cytometry and Cell Sorting to Characterize Native Human Bone Marrow Mesenchymal Stem Cells (MSC) . 121 Sally Boxall and Elena Jones 12 High Yield Recovery of Equine Mesenchymal Stem Cells from Umbilical Cord Matrix/Wharton’s Jelly Using a Semi-automated Process. 131 Timo Z. Nazari-Shafti, Ivone G. Bruno, Rudy Martinez, Michael E. Coleman, Eckhard U. Alt, and Scott R. McClure 13 Isolation and Functional Assessment of Cutaneous Stem Cells. 147 Yanne S. Doucet and David M. Owens vii viii Contents 14 Isolation of Adult Stem Cell Populations from the Human Cornea . 165 Matthew J. Branch, Wing-Yan Yu, Carl Sheridan, and Andrew Hopkinson 15 Advanced Imaging and Tissue Engineering of the Human Limbal Epithelial Stem Cell Niche. 179 Isobel Massie, Marc Dziasko, Alvena Kureshi, Hannah J. Levis, Louise Morgan, Michael Neale, Radhika Sheth, Victoria E. Tovell, Amanda J. Vernon, James L. Funderburgh, and Julie T. Daniels 16 Isolation and Characterization of Stem Cells in the Adult Mammalian Ovary . 203 Seema Parte, Hiren Patel, Kalpana Sriraman, and Deepa Bhartiya 17 Clonal Culture of Adult Mouse Lung Epithelial Stem/Progenitor Cells . 231 Jonathan L. McQualter and Ivan Bertoncello 18 Culture and Characterization of Mammary Cancer Stem Cells in Mammospheres. 243 Eleonora Piscitelli, Cinzia Cocola, Frank Rüdiger Thaden, Paride Pelucchi, Brian Gray, Giovanni Bertalot, Alberto Albertini, Rolland Reinbold, and Ileana Zucchi 19 Isolation and Culture of Primary Glioblastoma Cells from Human Tumor Specimens . 263 Sascha Seidel, Boyan K. Garvalov, and Till Acker Index . 277 Contributors TILL ACKER • Institute of Neuropathology , University of Giessen , Giessen , Germany ALBERTO ALBERTINI • Instituto di Technologie Biomediche , Segrate Milan , Italy ECKHARD U. ALT • Department of Surgery , Tulane University , New Orleans , LA , USA GIOVANNI BERTALOT • Azienda Ospedaliera Desenzano d/G , Leno , Italy IVAN BERTONCELLO • Lung Health Research Center , University of Melbourne , Melbourne , VIC , Australia DEEPA BHARTIYA • Stem Cell Department , National Institute for Research and Reproductive Health , Mumbai , India SALLY BOXALL • Leeds Institute of Rheumatic and Musculoskeletal Medicine, Leeds University , Leeds , UK MATTHEW J. BRANCH • Ophthalmology DCN , University of Nottingham , Nottingham , UK IVONE G. BRUNO • InGeneron, Inc. , Houston , TX , USA VINCENT M. CASSONE • Department of Biology, University of Kentucky , Lexington,