Neural Stem Cells : Methods and Protocols

Over the last decade, advances in neural stem cell research have raised the hope that one day cellular therapy will not only replace missing neurons, but also replenish absent chemical signals, metabolites, enzymes, neurotransmitters, or other missing or defective components from the diseased or injured brain. 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. Each is written by an investigator who has used the method extensively, and includes step-by-step instructions, tips on avoiding pitfalls, and invaluable notes that make all the difference to successful experimental outcomes. Comprehensive and easy-to-follow, Neural Stem Cells: Methods and Protocols provides a powerful synthesis of today's key in vitro and in vivo techniques to identify and characterize neural stem cells for research and for developing experimental therapeutics. Machine generated contents note: Prefacev Contributors xi PART I ISOLATION AND CULTURE OF NSCs 1 Neural Differentiation of Embryonic Stem Cells K. Sue O'Shea 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. Rao29 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 Roskams49 6 Production of Immortalized Human Neural Crest Stem Cells Seung U. Kim, Eiji Nakagawa, Kozo Hatori, Atsushi Nagai, Myung A. Lee, and Jung H. Bang55 7 Adult Rodent Spinal Cord Derived Neural Stem Cells: Isolation and Characterization Lamya S. Shihabuddin67 8 Preparation of Neural Progenitors from Bone Marrow and Umbilical Cord Blood Shijie Song and J. Sanchez-Ramos79 9 Seeding Neural Stem Cells on Scaffolds of PGA, PLA, and Their Copolymers Erin Lavik, Yang D. Teng, Evan Snyder, and Robert Langer 89 PART II CHARACTERIZATION OF NSCs IN VITRO A. CELLULAR TECHNIQUES 10 Analysis of Cell Generation in the Telencephalic Neuroepithelium Takao Takahashi, Verne S. Caviness, Jr., and Pradeep G. Bhide101 11 Clonal Analyses and Cryopreservation of Neural Stem Cell Cultures Angelo L. Vescovi, Rossella Galli, and Angela Gritti115 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. Prowse137 14 In Vitro Assays for Neural Stem Cell Differentiation Marcel M. Daadi 149 15 Electron Microscopy and Lac-Z Labeling Bela Kosaras and Evan Snyder157 B. ELECTROPHYSIOLOGICAL TECHNIQUES 16 Techniques for Studying the Electrophysiology of Neurons Derived from Neural Stem/Progenitor Cells David S. K. Magnuson and Dante J. Morassutti179 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. Gratsch197 19 Differential Display: Isolation of Novel Genes Theresa E. Gratsch213 20 Cell Labeling and Gene Misexpression by Electroporation Terence J. Van Raay and Michael R. Stark223 21 Gene Therapy Using Neural Stem Cells Luciano Conti and Elena Cattaneo 233 22 Modeling Brain Pathologies Using Neural Stem Cells Simonetta Siplone and Elena Cattaneo245 PART III UTILIZATION/CHARACTERIZATION OF NSCs IN VivO A. ENDOGENOUS POOLS OF 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 Musashil-Positive Cells in Human Normal and Neoplastic Neuroepithelial Tissues by Immunohistochemical Methods Yonehiro Kanemura, Shin-ichi Sakakibara, and Hideyuki Okano273 25 Identification of Newborn Cells by BrdU Labeling and Immunocytochemistry In Vivo Sanjay S. P. Magavi and Jeffrey D. Macklis283 26 Immunocytochemical Analysis of Neuronal Differentiation Sanjay S. P. Magavi and Jeffrey D. Macklis291 27 Neuroanatomical Tracing of Neuronal Projections with Fluoro-Gold Lisa A. Catapano, Sanjay S. P. Magavi, and Jeffrey D. Macklis299 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. Whittemore307 29 Optimizing Stem Cell Grafting into the CNS Scott R. Whittemore, Y. Ping Zhang, Christopher B. Shields, Dante J. Morassutti, and David S. K. Magnuson319 30 Vision-Guided Technique for Cell Transplantation and Injection of Active Molecules into Rat and Mouse Embryos Lorenzo M agrassi327 31 Transplantation into Neonatal Rat Brain as a Tool to Study Properties of Stem Cells Tanja Zigova and Mary B. Newman341 32 Routes of Stem Cell Administration in the Adult Rodent Alison E. Willing, Svitlana Garbuzova-Davis, Paul R. Sanberg, and Samuel Saporta357 Index3 7 5. Part I. Isolation And Culture Of Nscs -- 1. Neural Differentiation Of Embryonic Stem Cells / K. Sue O'shea -- 2. Production And Analysis Of Neurospheres From Acutely Dissociated And Postmortem Cns Specimens / Eric D. Laywell [and Others] -- 3. Isolation Of Stem And Precursor Cells From Fetal Tissue / Yuan Y. Wu, Tahmina Mujtaba, And Mahendra S. Rao -- 4. Olfactory Ensheathing Cells : Isolation And Culture From The Rat Olfactory Bulb / Susan C. Barnett And A. Jane Roskams -- 5. Culturing Olfactory Ensheathing Glia From The Mouse Olfactory Epithelium / Edmund Au And A. Jane Roskams -- 6. Production Of Immortalized Human Neural Crest Stem Cells / Seung U. Kim [and Others] -- 7. Adult Rodent Spinal Cord Derived Neural Stem Cells : Isolation And Characterization / Lamya S. Shihabuddin -- 8. Preparation Of Neural Progenitors From Bone Marrow And Umbilical Cord Blood / Shijie Song And J. Sanchez-ramos --^ 9. Seeding Neural Stem Cells On Scaffolds Of Pga, Pla, And Their Copolymers / Erin Lavik [and Others] -- Part Ii. Characterization Of 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 -- 11. Clonal Analyses And Cryopreservation Of Neural Stem Cell Cultures / Angelo L. Vescovi, Rossella Galli, And Angela Gritti -- 12. Assessing The Involvement Of Telomerase In Stem Cell Biology / Mark P. Mattson, Peisu Zhang, And Weiming Fu -- 13. Detection Of Telomerase Activity In Neural Cells / Karen R. Prowse -- 14. In Vitro Assays For Neural Stem Cell Differentiation / Marcel M. Daadi -- 15. Electron Microscopy And Lac-z Labeling / Bela Kosaras And Evan Snyder -- 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 --^ C. Molecular Techniques -- 17. Fluorescence In Situ Hybridization / Barbara A. Tate And Rachel L. Ostroff -- 18. Rt-pcr Analyses Of Differential Gene Expression In Es-derived Neural Stem Cells / Theresa E. Gratsch -- 19. Differential Display : Isolation Of Novel Genes / Theresa E. Gratsch -- 20. Cell Labeling And Gene Misexpression By Electroporation / Terence J. Van Raay And Michael R. Stark -- 21. Gene Therapy Using Neural Stem Cells / Luciano Conti And Elena Cattaneo -- 22. Modeling Brain Pathologies Using Neural Stem Cells / Simonetta Siplone And Elena Cattaneo -- Part Iii. Utilization/characterization Of Nscs In Vivo -- A. Endogenous Pools Of Stem/progenitor Cells -- 23. Activation And Differentiation Of Endogenous Neural Stem Cell Progeny In The Rat Parkinson Animal Model / Marcel M. Daadi --^ 24. Identification Of Musashil-positive Cells In Human Normal And Neoplastic Neuroepithelial Tissues By Immunohistochemical Methods / Yonehiro Kanemura, Shin-ichi Sakakibara, And Hideyuki Okano -- 25. Identification Of Newborn Cells By Brdu Labeling And Immunocytochemistry In Vivo -- Sanjay S.p. Magavi And Jeffrey D. Macklis283 -- 26. Immunocytochemical Analysis Of Neuronal Differentiation / Sanjay S.p. Magavi And Jeffrey D. Macklis -- 27. Neuroanatomical Tracing Of Neuronal Projections With Fluoro-gold / Lisa A. Catapano, Sanjay S.p. Magavi, And Jeffrey D. Macklis -- 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 -- 29. Optimizing Stem Cell Grafting Into The Cns / Scott R. Whittemore [and Others] -- 30. Vision-guided Technique For Cell Transplantation And Injection Of Active Molecules Into Rat And Mouse Embryos / Lorenzo Magrassi --^ 31. Transplantation Into Neonatal Rat Brain As A Tool To Study Properties Of Stem Cells / Tanja Zigova And Mary B. Newman -- 32. Routes Of Stem Cell Administration In The Adult Rodent / Alison E. Willing [and Others]. Edited By Tanja Zigova, Paul R. Sanberg, Juan R. Sanchez-ramos. Includes Bibliographical References And Index. Describes the most frequently used cellular, molecular, and electrophysiological methods to isolate, characterize, and utilize neural stem cells. This book features techniques, which help researchers identify endogenous stem cells as well as exogenous stem cells after transplantation in the brain. Differentiation of pluripotent embryonic stem (ES) cells into specific lineages is an important source of cells for implantation and gene delivery, as well as a useful model to study patterns of differentiation and gene expression during the very early development of the mammalian embryo (1). Univ. of South Florida, Tampa. Features a synthesis of latest techniques for isolation, identification, and characterization of neural stem cells. Includes cellular techniques, including cell cycle kinetics, telomerase assays, and electrophysiology. For basic and clinical researchers. Front Matter....Pages i-xiv Front Matter....Pages 1-1 Front Matter....Pages 3-13 Front Matter....Pages 15-26 Front Matter....Pages 29-39 Back Matter....Pages 41-47 ....Pages 49-53