Neural Stem Cells

In Neural Stem Cells: Methods and Protocols, internationally recognized experts from academic, clinical, and pharmaceutical laboratories describe in great detail the most frequently used cellular, molecular, and electrophysiological methods to isolate, characterize, and utilize neural stem cells. These readily reproducible techniques introduce the various sources of stem/progenitor cells, provide a wide range of conditions for their culture, and make it possible to define their properties in culture. Additional techniques are designed to help researchers identify endogenous stem cells as well as exogenous stem cells after transplantation in the brain. The protocols range from the simplest methods of isolation and characterization of neural cell properties to such very sophisticated methods as characterizing gene expression, telomerase assays, and cell cycle kinetics.

Preface

v

Contributors

xi

Part I Isolation and Culture of NSCs

Neural Differentiation of Embryonic Stem Cells

3

(12)

K. Sue O'Shea

Production and Analysis of Neurospheres from Acutely Dissociated and Postmortem CNS Specimens

15

(14)

Eric D. Laywell

Valery G. Kukekov

Oleg Suslov

Tong Zheng

Dennis A. Steindler

Isolation of Stem and Precursor Cells from Fetal Tissue

29

(12)

Yuan Y. Wu

Tahmina Mujtaba

Mahendra S. Rao

Olfactory Ensheathing Cells: Isolation and Culture from the Rat Olfactory Bulb

41

(8)

Susan C. Barnett

A. Jane Roskams

Culturing Olfactory Ensheathing Glia from the Mouse Olfactory Epithelium

49

(6)

Edmund Au

A. Jane Roskams

Production of Immortalized Human Neural Crest Stem Cells

55

(12)

Seung U. Kim

Eiji Nakagawa

Kozo Hatori

Atsushi Nagai

Myung A. Lee

Jung H. Bang

Adult Rodent Spinal Cord Derived Neural Stem Cells: Isolation and Characterization

67

(12)

Lamya S. Shihabuddin

Preparation of Neural Progenitors from Bone Marrow and Umbilical Cord Blood

79

(10)

Shijie Song

J. Sanchez-Ramos

Seeding Neural Stem Cells on Scaffolds of PGA, PLA, and Their Copolymers

89

(12)

Erin Lavik

Yang D. Teng

Evan Snyder

Robert Langer

Part II Characterization of NSCs in Vitro

A. Cellular Techniques

Analysis of Cell Generation in the Telencephalic Neuroepithelium

101

(14)

Takao Takahashi

Verne S. Caviness, Jr.

Pradeep G. Bhide

Clonal Analyses and Cryopreservation of Neural Stem Cell Cultures

115

(10)

Angelo L. Vescovi

Rossella Galli

Angela Gritti

Assessing the Involvement of Telomerase in Stem Cell Biology

125

(12)

Mark P. Mattson

Peisu Zhang

Weiming Fu

Detection of Telomerase Activity in Neural Cells

137

(12)

Karen R. Prowse

In Vitro Assays for Neural Stem Cell Differentiation

149

(8)

Marcel M. Daadi

Electron Microscopy and Lac-Z Labeling

157

(22)

Bela Kosaras

Evan Snyder

B. Electrophysiological Techniques

Techniques for Studying the Electrophysiology of Neurons Derived from Neural Stem/Progenitor Cells

179

(10)

David S. K. Magnuson

Dante J. Morassutti

C. Molecular Techniques

Fluorescence In Situ Hybridization

189

(8)

Barbara A. Tate

Rachel L. Ostroff

RT-PCR Analyses of Differential Gene Expression in ES-Derived Neural Stem Cells

197

(16)

Theresa E. Gratsch

Differential Display: Isolation of Novel Genes

213

(10)

Theresa E. Gratsch

Cell Labeling and Gene Misexpression by Electroporation

223

(10)

Terence J. Van Raay

Michael R. Stark

Gene Therapy Using Neural Stem Cells

233

(12)

Luciano Conti

Elena Cattaneo

Modeling Brain Pathologies Using Neural Stem Cells

245

(20)

Simonetta Sipione

Elena Cattaneo

Part III Utilization/Characterization of NSCS in Vivo

A. Endogenous Pools of Stem/progenitor Cells

Activation and Differentiation of Endogenous Neural Stem Cell Progeny in the Rat Parkinson Animal Model

265

(8)

Marcel M. Daadi

Identification of Musashi1-Positive Cells in Human Normal and Neoplastic Neuroepithelial Tissues by Immunohistochemical Methods

273

(10)

Yonehiro Kanemura

Shin-ichi Sakakibara

Hideyuki Okano

Identification of Newborn Cells by BrdU Labeling and Immunocytochemistry In Vivo

283

(8)

Sanjay S. P. Magavi

Jeffrey D. Macklis

Immunocytochemical Analysis of Neuronal Differentiation

291

(8)

Sanjay S. P. Magavi

Jeffrey D. Macklis

Neuroanatomical Tracing of Neuronal Projections with Fluoro-Gold

299

(8)

Lisa A. Catapano

Sanjay S. P. Magavi

Jeffrey D. Macklis

B. Transplantation

Labeling Stem Cells In Vitro for Identification of Their Differentiated Phenotypes After Grafting into the CNS

307

(12)

Qi-lin Cao

Stephen M. Onifer

Scott R. Whittemore

Optimizing Stem Cell Grafting into the CNS

319

(8)

Scott R. Whittemore

Y. Ping Zhang

Christopher B. Shields

Dante J. Morassutti

David S. K. Magnuson

Vision-Guided Technique for Cell Transplantation and Injection of Active Molecules into Rat and Mouse Embryos

327

(14)

Lorenzo Magrassi

Transplantation into Neonatal Rat Brain as a Tool to Study Properties of Stem Cells

341

(16)

Tanja Zigova

Mary B. Newman

Routes of Stem Cell Administration in the Adult Rodent

357

(18)

Alison E. Willing

Svitlana Garbuzova-Davis

Paul R. Sanberg

Samuel Saporta

Index

375

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