Schwann Cells

480 Pages·2018·16.143 MB·English

by  Paula V. Monje, Haesun A. Kim

Methods in
Molecular Biology 1739
Paula V. Monje
Haesun A. Kim Editors
Schwann
Cells
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
Schwann Cells
Methods and Protocols
Edited by
Paula V. Monje
Department of Neurological Surgery, University of Miami, Miami, FL, USA
Haesun A. Kim
Department of Biological Sciences, Rutgers University, Newark, NJ, USA
Editors
Paula V. Monje Haesun A. Kim
Department of Neurological Surgery Department of Biological Sciences
University of Miami Rutgers University
Miami, FL, USA Newark, NJ, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-7648-5 ISBN 978-1-4939-7649-2 (eBook)
https://doi.org/10.1007/978-1-4939-7649-2
Library of Congress Control Number: 2018931192
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Cover image: Dorsal root ganglion explant grown in vitro displaying Schwann cells (green) and neurons (red). Courtesy
of Dr. Felipe A. Court
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Preface
If we have normal cell types available in culture and if a nervous system disease stems from an
absence or deficiency of one of these cell types, then it is an obvious step to transfer cultured cells
into the diseased animal to attempt to correct the deficiency. Whereas at present this would not
seem practical for many types of neurons, it may be practical for supporting cells (e.g. Schwann
or glial).
–Richard P. Bunge (The changing uses of nerve tissue culture 1950–1975,
in The Nervous System, 1975)
The Schwann cell has traditionally been viewed as the ensheathing cell type of the periph-
eral nervous system associated with a multilamellar myelin sheath or a Remak bundle. This
somewhat static view of the Schwann cell has changed in recent years upon the continued
realization that besides their role in ensheathment and myelination, Schwann cells contrib-
ute to a variety of functions during nerve development, regeneration, and neurodegenera-
tive disease. Schwann cells have many more facets than initially expected. These fascinating
glial cells have an active role in immunomodulation, axon maintenance and regeneration,
synaptic transmission, cancer, and disease progression in many inherited and acquired neu-
ropathies. As wisely anticipated by Dr. Richard Bunge over 40 years ago, cultured Schwann
cells can serve as a therapeutic product for cell replacement strategies aimed at treating
central and peripheral nervous system injuries. Indeed, the safety of autologous human
Schwann cell transplantation is currently being tested as a strategy for spinal cord repair
through clinical trials in the United States. This is an unprecedented milestone for the clini-
cal testing of a glial cell product.
A new era in the study of Schwann cells has already emerged with the glia-neuron field
cross-talking with areas of more recent development such as bioengineering, materials sci-
ence, stem cell biology, and regenerative medicine. An exciting wave of knowledge on
Schwann cell biology in health and disease has resulted from the development of state-of-
the-art technologies in cell culture, genetic manipulation of cells in vitro and in vivo, and
high-resolution imaging techniques for live and fixed cells, tissues, and whole organisms.
The development of mammalian and nonmammalian animal models targeting Schwann
cells in peripheral nerves has greatly improved our understanding of myelination, Schwann
cell-axon communication, and mechanisms of disease at the cellular and molecular levels.
The availability of the Schwann cells themselves along with the products they secrete or
generate has motivated novel applications with potential for translation. The current feasi-
bility to generate Schwann cells “in a dish” from virtually any given type of cell has revolu-
tionized our concepts and greatly expanded the possibilities for basic and applied research.
The book Schwann Cells: Methods and Protocols intends to present an assortment of
traditional and emerging experimental procedures relevant to Schwann cell research. The
chapter collection consists of 31 chapters divided into four discrete parts. Part I contains
protocols for in vitro culture, purification, and characterization of primary Schwann cells
from diverse species and stages of nerve development. It also contains protocols to derive
v
vi Preface
cancer cell lines and create engineered Schwann cells from unconventional sources via
chemical conversion or induced differentiation. Parts II and III outline a wide range of
methodologies used to study Schwann cells within in vitro and in vivo systems, respectively,
relevant to the analysis of peripheral nerve development, cancer, axon degeneration/regen-
eration, and myelination. Part II encompasses protocols for assessing the myelinating
Schwann cell phenotype and the interactions of Schwann cells with neurons, nonneuronal
cell types, and the physical environment using organotypic and neuron-free cultures. Part
III focuses on the analysis and visualization of Schwann cells in dissected peripheral nerve
and human skin biopsies, as well as the whole organism in adult and larvae zebra fish, using
standard immunochemical approaches, high-resolution image analysis, and time-lapse
microscopy. Lastly, Part IV outlines protocols for Schwann cell production, collection,
labeling, and transplantation in the injured peripheral nerve and spinal cord of experimental
animals and human subjects.
We hope this selection provides a practical framework to aid both experienced and new
investigators make progress in their research endeavors involving Schwann cells. We are
immensely indebted to all of the scientists who contributed to this book by preparing the
chapters and sharing their unique experience in the development and use of these method-
ologies. We greatly appreciate their valuable time, insights, and dedication to the joint
effort of materializing this project. Our sincere thanks to John Walker first and foremost for
his recognition that the fascinating cell of Schwann merited an independent book in the
prestigious series Methods in Molecular Biology and for bringing this initiative to our atten-
tion, facilitating the preparation of the book, and mentoring us on editing the chapters.
Many thanks to all members of the Springer team who contributed to this project. A whole-
hearted special recognition to Patrick Marton for the expeditious assistance and support in
helping us navigate the miscellaneous aspects of editing and publishing.
To conclude, our understanding of Schwann cells, their function, and potential uses
relies primarily on the methods we use to approach their study. The scientific literature on
the subject of Schwann cells is currently advancing at an incredible pace. If this trend con-
tinues, a wave of groundbreaking research is likely to reshape our concepts sooner than
expected. We hope that readers find this comprehensive set of step-by-step protocols both
timely and useful in their experimental approaches while making a contribution to the
research and innovation that lies ahead in the Schwann cell field.
Miami, FL, USA Paula V. Monje
Newark, NJ, USA Haesun A. Kim
Contents
Preface .................................................................... v
Contributors ............................................................... xi
P I P P P C , C L
ART ROTOCOLS FOR REPARING RIMARY ULTURES ELL INES
E S C
AND NGINEERED CHWANN ELLS
1 Isolation of Schwann Cell Precursors from Rodents . . . . . . . . . . . . . . . . . . . . . 3
Rhona Mirsky and Kristjan R. Jessen
2 Preparation of Neonatal Rat Schwann Cells and Embryonic Dorsal
Root Ganglia Neurons for In Vitro Myelination Studies . . . . . . . . . . . . . . . . . . 17
Patrice Maurel
3 Isolation and Expansion of Schwann Cells from Transgenic Mouse Models . . . 39
Jihyun Kim and Haesun A. Kim
4 Isolation, Culture, and Cryopreservation of Adult Rodent Schwann
Cells Derived from Immediately Dissociated Teased Fibers . . . . . . . . . . . . . . . . 49
Natalia D. Andersen and Paula V. Monje
5 Detailed Protocols for the Isolation, Culture, Enrichment
and Immunostaining of Primary Human Schwann Cells . . . . . . . . . . . . . . . . . . 67
Tamara Weiss, Sabine Taschner-Mandl, Peter F. Ambros, and Inge M. Ambros
6 Magnetic-Activated Cell Sorting for the Fast and Efficient Separation
of Human and Rodent Schwann Cells from Mixed Cell Populations . . . . . . . . 87
Kristine M. Ravelo, Natalia D. Andersen, and Paula V. Monje
7 Culture and Expansion of Rodent and Porcine Schwann Cells
for Preclinical Animal Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Adriana E. Brooks, Gagani Athauda, Mary Bartlett Bunge, and Aisha Khan
8 Chemical Conversion of Human Fibroblasts into Functional Schwann Cells . . . 127
Eva C. Thoma
9 Derivation of Fate-Committed Schwann Cells from Bone Marrow
Stromal Cells of Adult Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Y.P. Tsui, Graham K. Shea, Y.S. Chan, and Daisy K.Y. Shum
10 Human Induced Pluripotent Stem Cell-Derived Sensory Neurons
for Fate Commitment of Bone Marrow Stromal Cell-Derived
Schwann Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Sa Cai, Daisy K.Y. Shum, and Ying-Shing Chan
11 Generation and Use of Merlin-Deficient Human Schwann Cells
for a High-Throughput Chemical Genomics Screening Assay . . . . . . . . . . . . . . 161
Alejandra M. Petrilli and Cristina Fernández-Valle
vii
viii Contents
P II P S S C B
ART ROTOCOLS FOR TUDYING CHWANN ELL EHAVIOR
M I V
AND YELINATION N ITRO
12 Lentiviral Transduction of Rat Schwann Cells and Dorsal Root
Ganglia Neurons for In Vitro Myelination Studies . . . . . . . . . . . . . . . . . . . . . . 177
Corey Heffernan and Patrice Maurel
13 Preservation, Sectioning, and Staining of Schwann Cell Cultures
for Transmission Electron Microscopy Analysis . . . . . . . . . . . . . . . . . . . . . . . . 195
Vania W. Almeida, Margaret L. Bates, and Mary Bartlett Bunge
14 Scalable Differentiation and Dedifferentiation Assays Using Neuron-Free
Schwann Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Paula V. Monje
15 The Pseudopod System for Axon-Glia Interactions: Stimulation
and Isolation of Schwann Cell Protrusions that Form in Response
to Axonal Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Yannick Poitelon and M. Laura Feltri
16 In Vitro Analysis of the Role of Schwann Cells on Axonal Degeneration
and Regeneration Using Sensory Neurons from Dorsal Root Ganglia . . . . . . . 255
Rodrigo López-Leal, Paula Diaz, and Felipe A. Court
17 A Culture Model to Study Neuron-Schwann Cell-Astrocyte Interactions . . . . . 269
Susana R. Cerqueira, Yee-Shuan Lee, and Mary Bartlett Bunge
18 Preparation of Matrices of Variable Stiffness for the Study
of Mechanotransduction in Schwann Cell Development . . . . . . . . . . . . . . . . . . 281
Mateusz M. Urbanski and Carmen V. Melendez-Vasquez
19 Purification of Exosomes from Primary Schwann Cells, RNA Extraction,
and Next-Generation Sequencing of Exosomal RNAs . . . . . . . . . . . . . . . . . . . 299
Cristian De Gregorio, Paula Díaz, Rodrigo López-Leal, Patricio Manque,
and Felipe A. Court
20 3D Cancer Migration Assay with Schwann Cells . . . . . . . . . . . . . . . . . . . . . . . . 317
Laura Fangmann, Steffen Teller, Pavel Stupakov, Helmut Friess,
Güralp O. Ceyhan, and Ihsan Ekin Demir
P III P S S C D
ART ROTOCOLS FOR TUDYING CHWANN ELL EVELOPMENT
M I V
AND YELINATION N IVO
21 Teased Fiber Preparation of Myelinated Nerve Fibers from Peripheral
Nerves for Vital Dye Staining and Immunofluorescence Analysis . . . . . . . . . . . 329
Alejandra Catenaccio and Felipe A. Court
22 Whole Mount Immunostaining on Mouse Sciatic Nerves to Visualize
Events of Peripheral Nerve Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Xin-Peng Dun and David B. Parkinson
23 The Use of Low Vacuum Scanning Electron Microscopy (LVSEM)
to Analyze Peripheral Nerve Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
Peter Bond and David B. Parkinson
Contents ix
24 Analysis of Myelinating Schwann Cells in Human Skin Biopsies . . . . . . . . . . . . 359
Mario A. Saporta and Renata de Moraes Maciel
25 Whole Mount In Situ Hybridization and Immunohistochemistry
for Zebrafish Larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Rebecca L. Cunningham and Kelly R. Monk
26 Transmission Electron Microscopy for Zebrafish Larvae and Adult
Lateral Line Nerve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Rebecca L. Cunningham and Kelly R. Monk
27 Live Imaging of Schwann Cell Development in Zebrafish . . . . . . . . . . . . . . . . . 401
Rebecca L. Cunningham and Kelly R. Monk
P IV P S C T
ART ROTOCOLS FOR CHWANN ELL RANSPLANTATION
28 Transplantation of Adult Rat Schwann Cells into the Injured
Spinal Cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Ying Dai and Caitlin E. Hill
29 Schwann Cell Transplantation Methods Using Biomaterials . . . . . . . . . . . . . . . 439
Christine D. Plant and Giles W. Plant
30 Viral Transduction of Schwann Cells for Peripheral Nerve Repair . . . . . . . . . . . 455
Christine D. Plant and Giles W. Plant
31 Intraspinal Delivery of Schwann Cells for Spinal Cord Injury . . . . . . . . . . . . . . 467
Andrea J. Santamaría, Juan P. Solano, Francisco D. Benavides,
and James D. Guest
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
Contributors
VANIA W. ALMEIDA • The Miami Project to Cure Paralysis, University of Miami Miller
School of Medicine, Miami, FL, USA
INGE M. AMBROS • Children’s Cancer Research Institute, Vienna, Austria
PETER F. AMBROS • Children’s Cancer Research Institute, Vienna, Austria
NATALIA D. ANDERSEN • The Miami Project to Cure Paralysis, Department of Neurological
Surgery, University of Miami Miller School of Medicine, Miami, FL, USA
GAGANI ATHAUDA • Department of Cellular Biology and Pharmacology, Herbert Wertheim
College of Medicine, Florida International University, Miami, FL, USA
MARGARET L. BATES • The Miami Project to Cure Paralysis, University of Miami Miller
School of Medicine, Miami, FL, USA
FRANCISCO D. BENAVIDES • The Miami Project to Cure Paralysis, University of Miami
Miller School of Medicine, Miami, FL, USA
PETER BOND • Electron Microscopy Centre, Plymouth University, Plymouth, Devon, UK
ADRIANA E. BROOKS • The Miami Project to Cure Paralysis and Department of
Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, USA;
Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine,
Miami, FL, USA
MARY BARTLETT BUNGE • The Miami Project to Cure Paralysis, University of Miami
Miller School of Medicine, Miami, FL, USA; Department of Cell Biology, University of
Miami Miller School of Medicine, Miami, FL, USA; Department of Neurological Surgery,
University of Miami Miller School of Medicine, Miami, FL, USA; Lois Pope LIFE Center,
Miami, FL, USA
SA CAI • School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of
Hong Kong, Hong Kong, China
ALEJANDRA CATENACCIO • Center for Integrative Biology, Faculty of Sciences, Universidad
Mayor, Santiago, Chile, FONDAP Center for Geroscience, Brain Health and Metabolism,
Santiago, Chile
SUSANA R. CERQUEIRA • The Miami Project to Cure Paralysis, Lois Pope LIFE Center,
University of Miami Miller School of Medicine, Miami, FL, USA
GÜRALP O. CEYHAN • Department of Surgery, Klinikum rechts der Isar, Technische
Universität München, Munich, Germany
YING-SHING CHAN • School of Biomedical Sciences, Li Ka Shing Faculty of Medicine,
The University of Hong Kong, Hong Kong, China
FELIPE A. COURT • Center for Integrative Biology, Faculty of Sciences, Universidad Mayor,
Santiago, Chile; FONDAP Center for Geroscience, Brain Health and Metabolism,
Santiago, Chile
REBECCA L. CUNNINGHAM • Department of Developmental Biology, Washington University
in St. Louis, St. Louis, MO, USA
YING DAI • Burke Medical Research Institute, White Plains, NY, USA; Weill Cornell
Medicine, Feil Family Brain and Mind Research Institute, New York, NY, USA
xi