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M E T H O D S I N M O L E C U L A R B I O L O G YTM
Neural Stem Cells
Methods and Protocols
Edited by
Tanja Zigova,
PhD
Department of Neurosurgery, University of South Florida
College of Medicine, Tampa, FL
Paul R. Sanberg, ,
PhD DSc
Department of Neurosurgery, University of South Florida
College of Medicine, Tampa, FL
and
Juan R. Sanchez-Ramos, ,
PhD MD
Department of Neurology, University of South Florida
College of Medicine, Tampa, FL
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Neural stem cells: methods and protocols / edited by Tanja Zigova, Juan R. Sanchez-Ramos,
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p. cm.—(Methods in molecular biology; 198)
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ISBN 0-89603-964-1 (alk. paper)
1. Neurons–Laboratory manuals. 2. Stem cells–Laboratory manuals. I. Zigova, Tanja. II. Sanberg,
Paul R.III. Sanchez-Ramos, Juan Raymond, 1945- IV. Series.
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Preface
Over the last decade, neural stem cell research has provided penetrating
insights into the plasticity and regenerative potential of the brain. Stem cells
have been isolated from embryonic as well as adult central nervous system
(CNS). Many non-CNS mammalian tissues also contain stem cells with a more
limited repertoire: the replacement of tissue-specific cells throughout the life-
time of the organism. Progress has been made in understanding fundamental
stem cell properties that depend on the interplay of extrinsic signaling factors
with intrinsic genetic programs within critical time frames. With this growing
knowledge, scientists have been able to change a neural stem cell’s fate. Un-
der certain conditions, neural stem cells have been induced to differentiate
into cells outside the expected neural lineage and conversely, stem cells from
nonneural tissue have been shown to transdifferentiate into cells with distinct
neural phenotypes.
At the moment, there is an accelerated effort to identify a readily avail-
able, socially acceptable stem cell that can be induced to proliferate in an undif-
ferentiated state and that can be manipulated at will to generate diverse cells
types. We are on the threshold of a great new therapeutic era of cellular therapy
that has as great, if not greater, potential as the current pharmacologic era, glo-
rified by antibiotics, anesthetics, pain killers, immunosuppressants, and psycho-
tropics. Cellular therapeutics carries the promise of replacing missing neurons,
but also may serve to replenish absent chemical signals, metabolites, enzymes,
neurotransmitters, or other missing or defective components from the diseased
or injured brain. Cellular therapies may provide the best vehicle for delivery of
genetic material for treatment of hereditary diseases.
Although a great deal of data has been gathered and insights have been
provided by researchers around the world, we are still in the dark about funda-
mental processes that determine cell fate or that maintain a cell’s“stemness.”
To take some of the mystery out of this field and to provide a practical guide
for the researcher, we have collected straightforward methods and protocols
used by outstanding scientists in the field. Our primary goal is to facilitate
research in neural stem cell biology by providing detailed protocols to both
stimulate and guide novices and veterans in this area.
v
vi Preface
We divided Neural Stem Cells: Methods and Protocols into three broad
sections. The first section, “Isolation and Culture of Neural Stem Cells” intro-
duces the reader to different sources of stem/progenitor cells and provides a
wide range of conditions for their selection, nourishment, growth and survival
in culture. The second section, “Characterization of Neural Stem Cells in vitro”
is a collection of the cellular, electrophysiological, and molecular techniques
required to define the characteristics of neural stem cells in culture. The third
section,“Utilization/Characterization of Neural Stem Cells in vivo,” is a col-
lection of techniques to identify and characterize endogenous stem cells as
well as exogenous stem cells after transplantation into the brain.
At this stage in Neural Stem Cell Biology , we have relied on the avail-
able state-of-the-art techniques to define the properties of these cells and to
test their inherent plasticity. We hope that this collection of methods and pro-
tocols, ranging from simple to sophisticated in complexity, will serve as a
handy guide for stem cell scientists. We expect that the user will develop even
more advanced techniques and strategies in this field. Like a good cookbook
full of recipes and cooking instructions, we are confident that experimenta-
tion with these procedures may generate even better results suited to the par-
ticular goals of the researcher.
We would like to acknowledge Professor John M. Walker who initially
suggested we put together this book and then later advised us throughout the
editorial process. We greatly appreciate the suggestions and encouragement
from Dr. Mahendra S. Rao. We especially thank Marcia McCall for her caring
assistance, attention to detail, and long hours invested into compiling this vol-
ume.
Tanja Zigova, PhD
Paul R. Sanberg, PhD, DSc
Juan R. Sanchez-Ramos, PhD, MD
Contents
Preface .............................................................................................................v
Contributors.....................................................................................................xi
PART I ISOLATIONAND CULTURE OF NSCS
1 Neural Differentiation of Embryonic Stem Cells
K. Sue O’Shea........................................................................................3
2 Production and Analysis of Neurospheres from Acutely
Dissociated and Postmortem CNS Specimens
Eric D. Laywell, Valery G. Kukekov, Oleg Suslov,
Tong Zheng, and Dennis A. Steindler...........................................15
3 Isolation of Stem and Precursor Cells from Fetal Tissue
Yuan Y. Wu, Tahmina Mujtaba, and Mahendra S. Rao...................29
4 Olfactory Ensheathing Cells: Isolation and Culture from the Rat
Olfactory Bulb
Susan C. Barnett and A. Jane Roskams...........................................41
5 Culturing Olfactory Ensheathing Glia from the Mouse
Olfactory Epithelium
Edmund Au and A. Jane Roskams....................................................49
6 Production of Immortalized Human Neural Crest Stem Cells
Seung U. Kim, Eiji Nakagawa, Kozo Hatori, Atsushi Nagai,
Myung A. Lee, and Jung H. Bang..................................................55
7 Adult Rodent Spinal Cord Derived Neural Stem Cells:
Isolation and Characterization
Lamya S. Shihabuddin........................................................................67
8 Preparation of Neural Progenitors from Bone Marrow
and Umbilical Cord Blood
Shijie Song and J. Sanchez-Ramos..................................................79
9 Seeding Neural Stem Cells on Scaffolds of PGA, PLA,
and Their Copolymers
Erin Lavik, Yang D. Teng, Evan Snyder, and Robert Langer.........89
vii
viii Contents
PART II CHARACTERIZATIONOF NSCS IN VITRO
A. CELLULAR TECHNIQUES
10 Analysis of Cell Generation in the Telencephalic Neuroepithelium
Takao Takahashi, Verne S. Caviness, Jr.,
and Pradeep G. Bhide...................................................................101
11 Clonal Analyses and Cryopreservation
of Neural Stem Cell Cultures
Angelo L. Vescovi, Rossella Galli, and Angela Gritti...................115
12 Assessing the Involvement of Telomerase in Stem Cell Biology
Mark P. Mattson, Peisu Zhang, and Weiming Fu...........................125
13 Detection of Telomerase Activity in Neural Cells
Karen R. Prowse................................................................................137
14 In Vitro Assays for Neural Stem Cell Differentiation
Marcel M. Daadi..................................................................................149
15 Electron Microscopy and Lac-Z Labeling
Bela Kosaras and Evan Snyder.......................................................157
B. ELECTROPHYSIOLOGICAL TECHNIQUES
16 Techniques for Studying the Electrophysiology of Neurons
Derived from Neural Stem/Progenitor Cells
David S. K. Magnuson and Dante J. Morassutti............................179
C. MOLECULAR TECHNIQUES
17 Fluorescence In Situ Hybridization
Barbara A. Tate and Rachel L. Ostroff............................................189
18 RT-PCR Analyses of Differential Gene Expression
in ES-Derived Neural Stem Cells
Theresa E. Gratsch............................................................................197
19 Differential Display: Isolation of Novel Genes
Theresa E. Gratsch............................................................................213
20 Cell Labeling and Gene Misexpression by Electroporation
Terence J. Van Raay and Michael R. Stark....................................223
21 Gene Therapy Using Neural Stem Cells
Luciano Conti and Elena Cattaneo..................................................233
22 Modeling Brain Pathologies Using Neural Stem Cells
Simonetta Sipione and Elena Cattaneo..........................................245
Contents ix
PART III UTILIZATION/CHARACTERIZATION OF NSCS IN VIVO
A. ENDOGENOUS POOLSOF STEM/PROGENITOR CELLS
23 Activation and Differentiation of Endogenous Neural Stem Cell
Progeny in the Rat Parkinson Animal Model
Marcel M. Daadi..................................................................................265
24 Identification of Musashi1-Positive Cells in Human Normal
and Neoplastic Neuroepithelial Tissues by
Immunohistochemical Methods
Yonehiro Kanemura, Shin-ichi Sakakibara,
and Hideyuki Okano......................................................................273
25 Identification of Newborn Cells by BrdU Labeling and
Immunocytochemistry In Vivo
Sanjay S. P. Magavi and Jeffrey D. Macklis...................................283
26 Immunocytochemical Analysis of Neuronal Differentiation
Sanjay S. P. Magavi and Jeffrey D. Macklis...................................291
27 Neuroanatomical Tracing of Neuronal Projections with Fluoro-Gold
Lisa A. Catapano, Sanjay S. P. Magavi,
and Jeffrey D. Macklis..................................................................299
B. TRANSPLANTATION
28 Labeling Stem Cells In Vitro for Identification of Their
Differentiated Phenotypes After Grafting into the CNS
Qi-lin Cao, Stephen M. Onifer, and Scott R. Whittemore.............307
29 Optimizing Stem Cell Grafting into the CNS
Scott R. Whittemore, Y. Ping Zhang, Christopher B. Shields,
Dante J. Morassutti, and David S. K. Magnuson.......................319
30 Vision-Guided Technique for Cell Transplantation and Injection
of Active Molecules into Rat and Mouse Embryos
Lorenzo Magrassi..............................................................................327
31 Transplantation into Neonatal Rat Brain as a Tool to Study
Properties of Stem Cells
Tanja Zigova and Mary B. Newman.................................................341
32 Routes of Stem Cell Administration in the Adult Rodent
Alison E. Willing, Svitlana Garbuzova-Davis,
Paul R. Sanberg, and Samuel Saporta.......................................357
Index............................................................................................................375
Contributors
EDMUND AU • Center for Molecular Medicine and Therapeutics, University
of British Columbia, Vancouver, British Columbia, Canada
JUNG H. BANG•Brain Disease Research Center, Ajou University School of
Medicine, Suwon, Korea
SUSAN C. BARNETT • CRC Beatson Laboratories, Garscube Estate, Glasgow,
G61 BD, Scotland
PRADEEP G. BHIDE • Department of Neurology, Massachusetts General
Hospital and Harvard Medical School, Boston, MA
QI-LIN CAO •Kentucky Spinal Cord Injury Research Center and Department
of Neurological Surgery, University of Louisville, School of Medicine,
Louisville, KY
LISA A. CATAPANO• Division of Neuroscience, Children’s Hospital,
Program in Neuroscience, Harvard Medical School, Boston, MA
ELENA CATTANEO•Department of Pharmacological Sciences and Center of
Excellence on Neurodegenerative Diseases, University of Milan, Milan, Italy
VERNE S. CAVINESS• Department of Neurology, Massachusetts General
Hospital and Harvard Medical School, Boston, MA
LUCIANO CONTI •Department of Pharmacological Sciences and Center of
Excellence on Neurodegenerative Diseases, University of Milan, Milan,
Italy, and Centre for Genome Research, University of Edinburgh,
Edinburgh, Scotland
MARCEL M. DAADI • Layton BioScience, Inc., Sunnyvale, CA
WEIMING FU • Laboratory of Neurosciences, National Institute on Aging
Gerontology Research Center, Baltimore, MD
ROSSELLA GALLI• Institute for Stem Cell Research, Ospedale “San
Raffaele,” Milan, Italy
SVITLANA GARBUZOVA-DAVIS•Center for Aging and Brain Repair, Department of
Neurosurgery, College of Medicine, University of South Florida, Tampa, FL
THERESA E. GRATSCH • Department of Cell and Developmental Biology,
University of Michigan Medical School, Ann Arbor, MI
ANGELA GRITTI •Institute for Stem Cell Research, Ospedale “San Raffaele,”
Milan, Italy
KOZO HATORI • Division of Neurology, Department of Medicine, University
of British Columbia, Vancouver, British Columbia, Canada
xi
