Neural Stem Cells: Methods and Protocols [1st ed.] 978-1-4939-9005-4, 978-1-4939-9007-8

Table of contents :
Front Matter ....Pages i-xiv
Generation of Neural Stem Cells from Induced Pluripotent Stem Cells (Marcel M. Daadi)....Pages 1-7
Making NSC and Neurons from Patient-Derived Tissue Samples (Odity Mukherjee, Shubhra Acharya, Mahendra Rao)....Pages 9-24
Generation of Definitive Neural Progenitor Cells from Human Pluripotent Stem Cells for Transplantation into Spinal Cord Injury (Mohamad Khazaei, Christopher S. Ahuja, Christopher E. Rodgers, Priscilla Chan, Michael G. Fehlings)....Pages 25-41
Derivation of Neural Stem Cells from Human Parthenogenetic Stem Cells (Rodolfo Gonzalez, Ibon Garitaonandia, Andrey Semechkin, Russell Kern)....Pages 43-57
Chemically Defined Neural Conversion of Human Pluripotent Stem Cells (Yu Chen, Carlos A. Tristan, Sunil K. Mallanna, Pinar Ormanoglu, Steven Titus, Anton Simeonov et al.)....Pages 59-72
In Vitro Functional Characterization of Human Neurons and Astrocytes Using Calcium Imaging and Electrophysiology (Marita Grønning Hansen, Daniel Tornero, Isaac Canals, Henrik Ahlenius, Zaal Kokaia)....Pages 73-88
Differentiation of Neural Stem Cells Derived from Induced Pluripotent Stem Cells into Dopaminergic Neurons (Marcel M. Daadi)....Pages 89-96
Midbrain Dopaminergic Neurons Differentiated from Human-Induced Pluripotent Stem Cells (Fabiano Araújo Tofoli, Ana Teresa Silva Semeano, Ágatha Oliveira-Giacomelli, Maria Carolina Bittencourt Gonçalves, Merari F. R. Ferrari, Lygia Veiga Pereira et al.)....Pages 97-118
Generating Neural Stem Cells from iPSCs with Dopaminergic Neurons Reporter Gene (Hyenjong Hong, Marcel M. Daadi)....Pages 119-128
Single-Cell Library Preparation of iPSC-Derived Neural Stem Cells (Jeffrey Kim, Marcel M. Daadi)....Pages 129-143
Bioinformatics Analysis of Single-Cell RNA-Seq Raw Data from iPSC-Derived Neural Stem Cells (Jeffrey Kim, Marcel M. Daadi)....Pages 145-159
Assay for Assessing Mitochondrial Function in iPSC-Derived Neural Stem Cells and Dopaminergic Neurons (Gourav Roy-Choudhury, Marcel M. Daadi)....Pages 161-173
Reference Transcriptome for Deriving Marmoset Induced Pluripotent Stem Cells (Guang Yang, Hyenjong Hong, April Torres, Kristen E. Malloy, Gourav Roy-Choudhury, Jeffrey Kim et al.)....Pages 175-186
Optimization of Differentiation of Nonhuman Primate Pluripotent Cells Using a Combinatorial Approach (Steven L. Farnsworth, Zhifang Qiu, Anuja Mishra, Peter J. Hornsby)....Pages 187-197
Isolation and Differentiation of Self-Renewable Neural Stem Cells from Marmoset-Induced Pluripotent Stem Cells (Hyenjong Hong, Gourav Roy-Choudhury, Jeffrey Kim, Marcel M. Daadi)....Pages 199-204
Lentiviral Infection of Mouse Bone Marrow Cells for Hematopoietic Stem Cell Transplantation (Cang Chen, Michael J. Guderyon, Guo Ge, Robert A. Clark, Senlin Li)....Pages 205-213
Central and Peripheral Secondary Cell Death Processes after Transient Global Ischemia in Nonhuman Primate Cerebellum and Heart (Jea-Young Lee, Roger Lin, Hung Nguyen, Eleonora Russo, M. Grant Liska, Trenton Lippert et al.)....Pages 215-225
Method for Stimulation of Hippocampal Neurogenesis by Transient Microneedle Insertion (Shijie Song, Xiaoyung Kong, Juan Sanchez-Ramos)....Pages 227-235
Back Matter ....Pages 237-239

Citation preview

Methods in Molecular Biology 1919

Marcel M. Daadi Editor

Neural Stem Cells Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

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

Neural Stem Cells Methods and Protocols

Edited by

Marcel M. Daadi Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA Department of Radiology, Research Imaging Institute, Cell Systems and Anatomy, Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

Editor Marcel M. Daadi Southwest National Primate Research Center Texas Biomedical Research Institute San Antonio, TX, USA Department of Radiology, Research Imaging Institute Cell Systems and Anatomy, Long School of Medicine University of Texas Health Science Center at San Antonio San Antonio, TX, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-9005-4 ISBN 978-1-4939-9007-8 (eBook) https://doi.org/10.1007/978-1-4939-9007-8 Library of Congress Control Number: 2018965603 © Springer Science+Business Media, LLC, part of Springer Nature 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Preface The field of neural stem cells is one of the most dynamic fields in modern neuroscience and regenerative medicine. It is an inspiring example of how basic and clinical research can fruitfully interact and synergize in a translational endeavor. After the isolation of neural stem cells from the brain in the early 1990s, the field has developed tremendously by exploiting multiple new sources of stem cells, by establishing innovative techniques to isolate and perpetuate neural stem cells, and by assessing their therapeutic potential in animal models of diseases. Since the discovery of somatic cell reprogramming techniques in 2006, stem cell research has entered a new exciting era of development and its scope has broadened to include evolving cellular and molecular approaches for cell isolation, growth, differentiation, tissue and organ modeling, and gene editing to derive therapeutic cells and to model or correct specific genetic diseases. This volume of Methods in Molecular Biology captures various technological advances to isolate, perpetuate, and characterize neural stem cells from various sources and to formulate them for delivery into animal models of neurological disorders. The chapters have been organized into four sections: (1) Definition and isolation of neural stem cells, (2) Characterization and differentiation, (3) Standards for defining cell populations, and (4) In vivo approaches in animal models. Chapter 1 addresses the cardinal properties that define a neural stem cell and its utility and rigor in answering basic biology questions, in the success of a drug discovery campaign, and in the efficacy of repairing diseased or injured tissue. The critical attributes of a neural stem cell population are a welldefined and a stable cellular composition, a consistent process of perpetuation that avoids drift in composition, and a stable profile of cellular differentiation or therapeutic activity. Chapter 2 discusses the recent developments in tissue reprogramming and human pluripotent stem cell differentiation into neural stem cells and neurons. The authors review the in vivo mechanisms underlying neural differentiation and methods to recapitulate these mechanisms in vitro. A map of transcription factors and morphogens of the developing brain is presented to guide derivation of regional and lineage-specific neural stem cells and differentiated progeny. Chapter 3 describes two protocols optimized for deriving definitive neural progenitor cells from human pluripotent stem cells and their differentiation into neurons, oligodendrocytes, and astrocytes. The authors also detail methods for generating the rodent model of spinal cord injury and for transplanting the neural stem cells. Chapter 4 describes a protocol for deriving neural stem cells from human parthenogenetic stem cells using a chemically defined method. The method developed is highly efficient with a reproducible differentiation profile. The neural stem cells derived under this technique are currently in clinical trials. Chapter 5 reports a robust, fast, small molecule-based procedure to generate neural stem cells from human pluripotent stem cells. This defined chemical composition may be used to dissect the mechanisms of neural lineage commitment and to develop systematic protocols that produce various cell types of the central nervous system at a large industrial scale. Chapter 6 goes into exquisite detail to rigorously characterize the functional properties of neural stem cell-derived neurons through calcium imaging and electrophysiological approaches. This is critical because neural stem cells and neurons may be generated by differentiation of embryonic and neural stem cells, by reprogramming somatic cells to pluripotency, or by direct conversion. The differentiation and maturation

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state of neurons depends on the source and method used. Thus, before considering using fully differentiated authentic neuronal populations for disease modeling or regenerative medicine, their functional properties and similarities to neurons of the central nervous system need to be defined. Here the authors employ calcium imaging and electrophysiology to define the functionality of neural stem cell-derived neurons. Chapter 7 describes a stepby-step technique to differentiate neural stem cells isolated from induced pluripotent stem cells into dopaminergic neurons. The method uses specific dopamine-inducing factors (DIF1). Chapter 8 reports a differential step-by-step method for generating midbrain dopaminergic neurons from human induced pluripotent stem cells cultured. This protocol yields populations of midbrain dopaminergic neurons with phenotypical and functional characteristics suitable for in vitro modeling, cell transplantation, and drug screening. Chapter 9 discusses genetic engineering neural stem cells with reporter genes specific to dopaminergic neurons, which enable studies to be conducted specifically on this neuronal population. The approach employs the CRISPR/Cas9 technique to insert the green fluorescent protein reporter gene to be driven by the tyrosine hydroxylase gene expression. Tyrosine hydroxylase is the rate-limiting enzyme for the biosynthesis of the dopamine and a reliable marker for dopaminergic neurons in vitro and in vivo. Single-cell RNA-Seq technology allows for the identification of heterogeneous cell populations, measurement of stochastic gene expressions, and identification of highly variable genes. Chapter 10 describes a protocol to capture and process a single-cell transcriptome using the C1 microfluidics system. The single-cell RNA-Seq library of neural stem cells is prepared using Fluidigm’s C1. This technology enables the identification of relevant pathways involved in development or in disease pathogenesis. Chapter 11 describes the bioinformatic data analysis that follows the generation of a neural stem cell RNA-Seq library (reported in Chapter 10). This chapter describes the bioinformatics analysis pipeline of single-cell sequencing data: raw sequencing data, quality checking samples, creating an index from a reference genome, aligning the sequences to indexing, and quantifying transcript abundances. The curated datasets are used for differential expression analysis and for analyses of populations and pathways. Mitochondrial bioenergetics is affected in many cell types in diseases and aging. Chapter 12 describes a rapid and reliable assay for assessing the mitochondrial function of neural stem cells used in investigating disease pathogenesis and drug discovery. The authors report a drug screening assay using the Seahorse XFe96 analyzer to measure mitochondrial functions in neural stem cells and neurons derived from induced pluripotent stem cells. Chapter 13 provides a stepby-step, quick and easy, transcriptomic assay to demonstrate pluripotency in nonhuman primate induced pluripotent stem cells. The assay is based on a reference set of genes and an expression signature used as a standard to test for pluripotency in any induced pluripotent stem cell line. This method enhances quality of cell lines, rigor, and reproducibility between various laboratories working on the same cell lines. Chapter 14 presents an iterative approach to systematically test the effects of various combinations of small molecules and biological factors on the differentiation of nonhuman primate pluripotent stem cells toward neural lineages. The approach uses a cyclical process, with multiple rounds to select the optimal combination of factors and concentrations. Chapter 15 discusses an approach for isolating self-renewable neural stem cells from marmoset induced pluripotent stem cells. Although nonhuman primate pluripotent stem cells may require different methodologies than those used for expansion or differentiation of human stem cells, similar methodologies work most of the time, as reported here. Chapter 16 describes a methodology to deliver growth factors to the central nervous system using hematopoietic stem cells. The stem cells are transduced with a lentiviral vector expressing the therapeutic gene under lineage-specific

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promoters, such as macrophages. After bone marrow engraftment, genetically engineered hematopoietic stem cells give rise to macrophages that penetrate the brain and migrate to areas of pathology. Chapter 17 addresses the in vivo model of ischemic brain injury and highlights the existence of other areas distant from the site of injury. This is important when considering neural stem cell therapy for stroke. Chapter 18 describes a stereotaxic approach to inject cells into the brain, specifically the hippocampus, and the effects of the needle injection on neurogenesis in the same brain region. Altogether these chapters address practical issues and provide rich technical information to enable scientists to use neural stem cells in their research programs. I wish to express my deep appreciation and gratitude to all the authors for contributing their work to this book. I would like to thank the editors at Springer for their support throughout the lengthy process of bringing the idea for this book to fruition. San Antonio, TX, USA

Marcel M. Daadi

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v xi

1 Generation of Neural Stem Cells from Induced Pluripotent Stem Cells . . . . . . . . 1 Marcel M. Daadi 2 Making NSC and Neurons from Patient-Derived Tissue Samples . . . . . . . . . . . . . 9 Odity Mukherjee, Shubhra Acharya, and Mahendra Rao 3 Generation of Definitive Neural Progenitor Cells from Human Pluripotent Stem Cells for Transplantation into Spinal Cord Injury . . . . . . . . . . . 25 Mohamad Khazaei, Christopher S. Ahuja, Christopher E. Rodgers, Priscilla Chan, and Michael G. Fehlings 4 Derivation of Neural Stem Cells from Human Parthenogenetic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Rodolfo Gonzalez, Ibon Garitaonandia, Andrey Semechkin, and Russell Kern 5 Chemically Defined Neural Conversion of Human Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Yu Chen, Carlos A. Tristan, Sunil K. Mallanna, Pinar Ormanoglu, Steven Titus, Anton Simeonov, and Ilyas Singec¸ 6 In Vitro Functional Characterization of Human Neurons and Astrocytes Using Calcium Imaging and Electrophysiology . . . . . . . . . . . . . . . 73 Marita Grønning Hansen, Daniel Tornero, Isaac Canals, Henrik Ahlenius, and Zaal Kokaia 7 Differentiation of Neural Stem Cells Derived from Induced Pluripotent Stem Cells into Dopaminergic Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Marcel M. Daadi 8 Midbrain Dopaminergic Neurons Differentiated from Human-Induced Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Fabiano Arau´jo Tofoli, Ana Teresa Silva Semeano, ´ gatha Oliveira-Giacomelli, Maria Carolina Bittencourt Gonc¸alves, A Merari F. R. Ferrari, Lygia Veiga Pereira, and Henning Ulrich 9 Generating Neural Stem Cells from iPSCs with Dopaminergic Neurons Reporter Gene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Hyenjong Hong and Marcel M. Daadi 10 Single-Cell Library Preparation of iPSC-Derived Neural Stem Cells . . . . . . . . . . . 129 Jeffrey Kim and Marcel M. Daadi 11 Bioinformatics Analysis of Single-Cell RNA-Seq Raw Data from iPSC-Derived Neural Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Jeffrey Kim and Marcel M. Daadi

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Contents

Assay for Assessing Mitochondrial Function in iPSC-Derived Neural Stem Cells and Dopaminergic Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gourav Roy-Choudhury and Marcel M. Daadi Reference Transcriptome for Deriving Marmoset Induced Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Guang Yang, Hyenjong Hong, April Torres, Kristen E. Malloy, Gourav Roy-Choudhury, Jeffrey Kim, and Marcel M. Daadi Optimization of Differentiation of Nonhuman Primate Pluripotent Cells Using a Combinatorial Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Steven L. Farnsworth, Zhifang Qiu, Anuja Mishra, and Peter J. Hornsby Isolation and Differentiation of Self-Renewable Neural Stem Cells from Marmoset-Induced Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . Hyenjong Hong, Gourav Roy-Choudhury, Jeffrey Kim, and Marcel M. Daadi Lentiviral Infection of Mouse Bone Marrow Cells for Hematopoietic Stem Cell Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cang Chen, Michael J. Guderyon, Guo Ge, Robert A. Clark, and Senlin Li Central and Peripheral Secondary Cell Death Processes after Transient Global Ischemia in Nonhuman Primate Cerebellum and Heart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jea-Young Lee, Roger Lin, Hung Nguyen, Eleonora Russo, M. Grant Liska, Trenton Lippert, Yuji Kaneko, and Cesar V. Borlongan Method for Stimulation of Hippocampal Neurogenesis by Transient Microneedle Insertion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shijie Song, Xiaoyung Kong, and Juan Sanchez-Ramos

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors SHUBHRA ACHARYA  Institute of Stem Cells and Regenerative Medicine (inStem), Bangalore, India HENRIK AHLENIUS  Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Lund Stem Cell Center, Stem Cells, Aging and Neurodegeneration Group, Lund University, Lund, Sweden CHRISTOPHER S. AHUJA  Division of Genetics and Development, Krembil Research Institute, Toronto, ON, Canada; Institute of Medical Science, University of Toronto, Toronto, ON, Canada; Division of Neurosurgery, University of Toronto, Toronto, ON, Canada CESAR V. BORLONGAN  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA ISAAC CANALS  Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Lund Stem Cell Center, Stem Cells, Aging and Neurodegeneration Group, Lund University, Lund, Sweden PRISCILLA CHAN  Division of Genetics and Development, Krembil Research Institute, Toronto, ON, Canada; Institute of Medical Science, University of Toronto, Toronto, ON, Canada CANG CHEN  Department of Medicine, UT Health Science Center at San Antonio, San Antonio, TX, USA YU CHEN  Stem Cell Translation Laboratory (SCTL), Division of Pre-Clinical Innovation, NIH National Center for Advancing Translational Sciences (NCATS), NIH Regenerative Medicine Program, Rockville, MD, USA ROBERT A. CLARK  Department of Medicine, UT Health Science Center at San Antonio, San Antonio, TX, USA; Research and Development Service, Audie L. Murphy VA Hospital, San Antonio, TX, USA MARCEL M. DAADI  Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA; Department of Radiology, Research Imaging Institute, Cell Systems and Anatomy, Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA STEVEN L. FARNSWORTH  Department of Cellular and Integrative Physiology and Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, TX, USA MICHAEL G. FEHLINGS  Division of Genetics and Development, Krembil Research Institute, Toronto, ON, Canada; Institute of Medical Science, University of Toronto, Toronto, ON, Canada; Division of Neurosurgery, University of Toronto, Toronto, ON, Canada; Spinal Program, Toronto Western Hospital, University Health Network, Toronto, ON, Canada; Faculty of Medicine, University of Toronto, Toronto, ON, Canada MERARI F. R. FERRARI  Institute of Biosciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil IBON GARITAONANDIA  International Stem Cell Corporation, Carlsbad, CA, USA GUO GE  Department of Medicine, UT Health Science Center at San Antonio, San Antonio, TX, USA MARIA CAROLINA BITTENCOURT GONC¸ALVES  Department of Neurology and Neuroscience, Medical School, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil

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Contributors

RODOLFO GONZALEZ  International Stem Cell Corporation, Carlsbad, CA, USA MICHAEL J. GUDERYON  Department of Medicine, UT Health Science Center at San Antonio, San Antonio, TX, USA MARITA GRØNNING HANSEN  Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Lund Stem Cell Center, Laboratory of Stem Cells and Restorative Neurology, Lund University, Ska˚ne University Hospital, Lund, Sweden HYENJONG HONG  Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA PETER J. HORNSBY  Department of Cellular and Integrative Physiology and Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, TX, USA YUJI KANEKO  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA RUSSELL KERN  International Stem Cell Corporation, Carlsbad, CA, USA MOHAMAD KHAZAEI  Division of Genetics and Development, Krembil Research Institute, Toronto, ON, Canada JEFFREY KIM  Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA ZAAL KOKAIA  Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Lund Stem Cell Center, Laboratory of Stem Cells and Restorative Neurology, Lund University, Ska˚ne University Hospital, Lund, Sweden XIAOYUNG KONG  Department of Neurology and Neurosurgery, University of South Florida, Tampa, FL, USA; College of Medicine Neurology, University of South Florida, Tampa, FL, USA; College of Medicine Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA JEA-YOUNG LEE  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA SENLIN LI  Department of Medicine, UT Health Science Center at San Antonio, San Antonio, TX, USA; Research and Development Service, Audie L. Murphy VA Hospital, San Antonio, TX, USA; Department of Pharmacology, University of Texas Health San Antonio, San Antonio, TX, USA ROGER LIN  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA TRENTON LIPPERT  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA M. GRANT LISKA  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA

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SUNIL K. MALLANNA  Stem Cell Translation Laboratory (SCTL), Division of Pre-Clinical Innovation, NIH National Center for Advancing Translational Sciences (NCATS), NIH Regenerative Medicine Program, Rockville, MD, USA KRISTEN E. MALLOY  Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA; Department of Radiology, Research Imaging Institute, Cell Systems and Anatomy, Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA ANUJA MISHRA  Department of Cellular and Integrative Physiology, Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, TX, USA ODITY MUKHERJEE  Institute of Stem Cells and Regenerative Medicine (inStem), Bangalore, India HUNG NGUYEN  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA ´ AGATHA OLIVEIRA-GIACOMELLI  Department of Biochemistry, Institute of Chemistry, University of Sa˜o Paulo, Sa˜o Paulo, Brazil PINAR ORMANOGLU  Stem Cell Translation Laboratory (SCTL), Division of Pre-Clinical Innovation, NIH National Center for Advancing Translational Sciences (NCATS), NIH Regenerative Medicine Program, Rockville, MD, USA ZHIFANG QIU  Department of Cellular and Integrative Physiology and Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, TX, USA MAHENDRA RAO  Institute of Stem Cells and Regenerative Medicine (inStem), Bangalore, India; Q Therapeutics, Salt Lake City, UT, USA CHRISTOPHER E. RODGERS  Division of Genetics and Development, Krembil Research Institute, Toronto, ON, Canada; Institute of Medical Science, University of Toronto, Toronto, ON, Canada GOURAV ROY-CHOUDHURY  Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA ELEONORA RUSSO  Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA JUAN SANCHEZ-RAMOS  Department of Neurology and Neurosurgery, University of South Florida, Tampa, FL, USA; Department of Neurology, James A. Haley VAH Research Service, University of South Florida, Tampa, FL, USA ANA TERESA SILVA SEMEANO  Department of Biochemistry, Institute of Chemistry, University of Sa˜o Paulo, Sa˜o Paulo, Brazil ANDREY SEMECHKIN  International Stem Cell Corporation, Carlsbad, CA, USA ANTON SIMEONOV  Stem Cell Translation Laboratory (SCTL), Division of Pre-Clinical Innovation, NIH National Center for Advancing Translational Sciences (NCATS), NIH Regenerative Medicine Program, Rockville, MD, USA ILYAS SINGEC¸  Stem Cell Translation Laboratory (SCTL), Division of Pre-Clinical Innovation, NIH National Center for Advancing Translational Sciences (NCATS), NIH Regenerative Medicine Program, Rockville, MD, USA SHIJIE SONG  Department of Neurology and Neurosurgery, University of South Florida, Tampa, FL, USA; Department of Neurology, James A. Haley VAH Research Service, University of South Florida, Tampa, FL, USA

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Contributors

STEVEN TITUS  Stem Cell Translation Laboratory (SCTL), Division of Pre-Clinical Innovation, NIH National Center for Advancing Translational Sciences (NCATS), NIH Regenerative Medicine Program, Rockville, MD, USA FABIANO ARAU´JO TOFOLI  Institute of Biosciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil DANIEL TORNERO  Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Lund Stem Cell Center, Laboratory of Stem Cells and Restorative Neurology, Lund University, Ska˚ne University Hospital, Lund, Sweden APRIL TORRES  Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA CARLOS A. TRISTAN  Stem Cell Translation Laboratory (SCTL), Division of Pre-Clinical Innovation, NIH National Center for Advancing Translational Sciences (NCATS), NIH Regenerative Medicine Program, Rockville, MD, USA HENNING ULRICH  Department of Biochemistry, Institute of Chemistry, University of Sa˜o Paulo, Sa˜o Paulo, Brazil LYGIA VEIGA PEREIRA  Institute of Biosciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil GUANG YANG  Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA

Chapter 1 Generation of Neural Stem Cells from Induced Pluripotent Stem Cells Marcel M. Daadi Abstract Neural stem cells (NSCs) are defined by three necessary but not sufficient criteria: (1) self-renewable, (2) ability to generate a large number of progeny, and (3) ability to differentiate into the principal central nervous system (CNS) cell types, neurons, astrocytes, and oligodendrocytes. There are various approaches to derive neural lineages from pluripotent stem cells. It is well recognized that the chosen method of NSC derivation is critical to answering the basic biology question under investigation, to the success rate in drug discovery and to the efficacy of the therapeutic cells intended for repairing the CNS. There are three critical attributes of NSCs: (1) well-defined and stable cellular composition, (2) consistent process of perpetuation that avoids drift in composition, and (3) stable phenotype or therapeutic activity of the NSCs or their differentiated progeny. Over the past decades, we have been continuously developing consistent processes for generating stable, multipotent self-renewable NSCs from various sources. In this chapter, we report a method to generate NSCs from induced pluripotent stem cells. Key words Self-renewal, iPSCs, Multipotent neural stem cells

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Introduction The three criteria that define neural stem cells (NSCs) are selfrenewal, generation of a large number of progeny, and differentiation into the principal central nervous system (CNS) cell types: neurons, astrocytes, and oligodendrocytes. NSCs can be derived from brain tissue, adult or developing [1, 2], or from pluripotent human embryonic stem cells [3]. However, due to their practical and versatile applications, especially in disease modeling, induced pluripotent stem cells (iPSCs) are often seen as a more attractive source of NSCs. There are two main approaches to derive neural lineages from pluripotent stem cells: Process A) a cyclic indefinite process consisting of regular passaging the NSCs, isolated from iPSCs, to perpetuate them without the return to the parental iPSCs and Process B) a linear and finite process started by differentiating iPSCs into neural lineage over an extended period of time

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Marcel M. Daadi

and which requires the repetitive use of the parental iPSCs. A previous study reported that Process B may lead to heterogeneous populations of neural and non-neural cells that may be considered as impurities and therefore would impact the experimental outcome [4]. In addition, Process B does not offer the opportunity to confirm whether the neural cells generated are actually NSCs, since the three criteria are not demonstrated. In this chapter, we describe Process A for generating NSCs from iPSCs.

2 2.1

Materials Equipment

1. Cell culture incubator (Nuaire, Plymouth, MN, USA). 2. Phase-contrast microscope (Zeiss, Oberkochen, Germany). 3. Centrifuge (Eppendorf, Hamburg, Germany). 4. T-25 tissue culture flask (Corning, Oneonta, NY, USA). 5. T-75 tissue culture flask (Corning, Oneonta, NY, USA). 6. Cell lifter (Fisher scientific, Pittsburgh, PA, USA). 7. Glass pipettes (Fisher scientific, Pittsburgh, PA, USA). 8. Centrifuge tubes (15 mL and 50 mL) (Corning, Oneonta, NY, USA). 9. Syringe filters (Corning, Oneonta, NY, USA). 10. Syringes (10, 20, and 60 mL) (BD, Franklin Lakes, NJ, USA). 11. Pipettes (2–25 mL) (Fisher scientific, Pittsburgh, PA, USA). 12. Pipette aids (10–1000 μL) (Eppendorf, Hamburg, Germany). 13. Pipette tips (10–1000 μL) (Accuflow, E&K Scientific, Santa Clara, CA, USA). 14. Water bath (Fisher scientific, Pittsburgh, PA, USA). 15. Hemocytometer (Hausser Scientific, Horsham, PA, USA) or automated cell counter (Countess, Invitrogen, Carlsbad, CA, USA).

2.2

Reagents

1. NN1 media (NeoNeuron, San Antonio, TX, USA). 2. DIF1 (NeoNeuron, San Antonio, TX, USA). 3. Basic fibroblast growth factor (Stemgent, Cambridge, MA, USA). 4. Epidermal growth factor (EGF, EMD Millipore, Burlington, MA, USA). 5. Poly-L-ornithine hydrobromide (Sigma-Aldrich, St. Louis, MO, USA). 6. Fetal bovine serum (FBS, GE, Logan, UT, USA). 7. Ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA).

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8. Accutase (Gibco, Life Technologies, NY, USA). 9. Trypsin neutralizer (Gibco, Life Technologies, NY, USA). 10. Tris base (Fisher BioReagents, Pittsburgh, PA, USA). 11. Phosphate-buffered saline (PBS, Gibco, Life Technologies, NY, USA). 12. Double-distilled water (Gibco, Life Technologies, NY, USA).

3

Methods

3.1 Preparation of Reagents and Media

Prepare all the reagents under sterile conditions in a horizontal laminar flow hood. 1. Preparation of 10 mM Tris (25 mL): Dissolve 30.35 mg of Tris base (F.Wt: 121.4) in 15 mL of double-distilled water, and adjust the pH to 7.6. Make up the volume to 25 mL and filter sterilize. Store at 4
C (see Note 1). 2. Preparation of basic fibroblast growth factor stock solution: Briefly centrifuge the tube and reconstitute the bFGF in 2.5 mL of 10 mM Tris solution (pH 7.6) to prepare a 20 μg/mL stock solution. Aliquot and store at 20
C. 3. Preparation of epidermal growth factor stock solution: Briefly centrifuge the tube, and reconstitute the EGF in 5 mL of double-distilled water to prepare a stock solution of 100 μg/ mL. Aliquot and store at 20
C. 4. Preparation of neural stem cell (NSC) media (50 mL): Add 50 μL of bFGF (20 μg/mL) and 10 μL of EGF (100 μg/mL) to 50 mL of NN1 media (NeoNeuron) to achieve a final concentration of 20 ng/mL of bFGF and EGF, respectively. Filter sterilize and store at 4
C. 5. Preparation of NSC freezing media (50 mL): Add 25 mL of NN1 media, 20 mL of FBS, and 5 mL DMSO, and mix. Filter sterilize and store at 4
C.

3.2 Isolation of NSCs from iPSCs

1. Culture and expand iPSCs in a 6-well plate until they are 80–90% confluent. 2. On the day of the experiment, prewarm the NSC media in a 37
C water bath. 3. Non-enzymatically detach the iPSC colonies using a cell lifter. Take care not to dissociate colonies into single cells. 4. Carefully collect the colonies from the 6-well plate using a 5 mL pipette, and transfer them to a 15 mL centrifuge tube. Do not triturate the cells. 5. Centrifuge at 1000 RPM (200  g) for 5 min.

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Fig. 1 Growth rate of self-renewable neural stem cells derived from iPSCs. (a) Photograph showing threedimensional organoid morphology of neural stem cells (NSCs) or “neurospheres” isolated from induced pluripotent stem cells (iPSCs) grown in a suspension culture. (b) Photograph showing two-dimensional morphology of NSCs derived from iPSCs grown as monolayer. (c) Photograph showing three-dimensional organoid morphology of NSCs isolated from cadaveric brain tissue and grown in a suspension culture “neurospheres.” (d) Graph showing the long-term growth stability of NSCs isolated from iPSCs over a period of 90 days

6. Using a vacuum-aided suction, carefully remove the media without disturbing the pellet using a glass pipette. 7. Resuspend the pellet in fresh 5 mL of NSC media. 8. Centrifuge at 1000 RPM (200  g) for 5 min. 9. Resuspend in 8 mL of NSC media and plate in a T-25 flask. 10. Three days after plating or when the media turns yellow, replace old media with fresh NSC media (NN1 þ 20 ng/mL bFGF þ2-ng/mL EGF) (see Note 2). 11. Change the media every 3–4 days as required. 12. To change media carefully remove the supernatant by using vacuum suction without disturbing the pellet. 13. Resuspend the pellet in fresh NSC media, and replate it into a T-25 flask. 14. Change the media every 3–4 days. 15. By the end of 2 weeks, NSCs can be seen to form neurospheres (Fig. 1).

iPSC-Derived Self-Renewable Neural Stem Cells

3.3 Initial Expansion of iPSC-Derived NSCs

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1. Prewarm NSC media, Accutase, and trypsin neutralizer in a 37
C water bath. 2. Fourteen days after the initial plating, collect all the floating neurospheres from the T-25 flask into a 15 mL tube, and centrifuge at 1000 RPM (200  g) for 5 min (Fig. 1). 3. Carefully remove the supernatant by using vacuum suction without disturbing the pellet. 4. Resuspend the pellet in 1 mL Accutase and incubate for 3 min at 37
C. 5. Inhibit the action of Accutase by adding 1 mL of trypsin neutralizer, and gently triturate 8–10 times to dissociate the cells into single-cell suspension (see Note 3). 6. Add 8 mL of culture media and centrifuge at 1000 RPM (200  g) for 5 min. 7. Remove the supernatant, and resuspend the cells in 3–5 mL of culture media. 8. Count the cells using either hemocytometer or automate cell counter using trypan blue exclusion. Calculate percentage viability of the cells, and record in the cell proliferation logbook. 9. Plate the cells at a density of 2  106 cells per T-25 tissue culture flask in 8 mL of NSC media. 10. Change media every 3–4 days. 11. Passage cells on the seventh day of the culture.

3.4 Scaling Up the Culture of iPSCDerived NSCs

After two or three passages, the NSC cultures can be scaled up to T-75 flasks for use in experiments or prepared for cryostorage (see Subheading 3.5). 1. Prewarm NSC media, Accutase, and trypsin neutralizer in a 37
C water bath. 2. On day 7 of NSC culture, collect all the neurospheres from the T-25 flask into a 15 mL tube and centrifuge at 1000 RPM (200  g) for 5 min. 3. Carefully remove the supernatant by using vacuum suction without disturbing the pellet. 4. Resuspend the pellet in 1 mL Accutase and incubate for 3 min at 37
C. 5. Inhibit the action of Accutase by adding 1 mL of trypsin neutralizer, and gently triturate 8–10 times to dissociate the cells into single-cell suspension. 6. Add 8 mL of culture media and centrifuge at 1000 RPM (200  g) for 5 min. 7. Remove the supernatant and resuspend the cells in 3–5 mL of culture media.

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8. Count the cells using either hemocytometer or automate cell counter using trypan blue exclusion. Calculate percentage viability of the cells, and record in the cell proliferation logbook. 9. Plate the cells at a density of 4  106 cells per T-75 tissue culture flask in 20 mL of NSC media. 10. Change media every 3–4 days. 11. Passage the cells every 7 days. 3.5 Freezing NSCs for Storage

1. After 3 days in vitro, collect the neurospheres from T-75 tissue culture flask into a 50 mL centrifuge tube. 2. Centrifuge the neurospheres at 1000 RPM (200  g) for 5 min. 3. Remove the supernatant and resuspend the neurospheres in 3 mL of freezing media. 4. Aliquot 1 mL NSC suspension in cryovials and store at 80
C overnight. 5. On day 4, transfer the vials to a liquid nitrogen tank and record cell type, passage, and date of freezing in the logbook.

4

Notes 1. Prepare all the solutions and reagents in double-distilled water or ultrapure water (18 MΩ cm at 25
C). 2. Cell death can be seen during early stages of NSC isolation from iPSCs. Carefully monitor the media color and change the media as required. 3. The trituration should be gentle to dissociate the cells without damaging the cellular integrity. Too severe trituration can reduce viability of the NSCs and affect cellular composition.

Acknowledgment This work was supported by NeoNeuron LLC. Disclosures: Dr. Marcel M. Daadi is founder of the biotech company NeoNeuron.

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References 1. Daadi MM, Weiss S (1999) Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain. J Neurosci 19(11):4484–4497 2. Reynolds BA, Weiss S (1996) Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic precursor is a stem cell. Dev Biol 175:1–13 3. Daadi MM, Maag AL, Steinberg GK (2008) Adherent self-renewable human embryonic

stem cell-derived neural stem cell line: functional engraftment in experimental stroke model. PLoS One 3(2):e1644 4. Brederlau A et al (2006) Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma formation. Stem Cells 24(6):1433–1440

Chapter 2 Making NSC and Neurons from Patient-Derived Tissue Samples Odity Mukherjee, Shubhra Acharya, and Mahendra Rao Abstract The human brain and mechanisms underlying its functioning has been a field of intense research due to its complexity, inaccessibility, and the large numbers of debilitating disorders affecting this organ. Model organisms have provided great insight into the functioning of the mammalian brain; however, there exist many features unique to humans which need detailed understanding. In this context, human pluripotent stem cells (HPSCs) have emerged as a promising resource. In the developing brain, cortical diversification is achieved by neural stem cells/neural progenitor cells (NSCs/NPCs) by altering its potency (from multipotent to unipotent) and differentiation capacity (from neurogenesis to gliogenesis). Recent development in tissue reprogramming allows for derivation of NSCs/ NPCs from either healthy control subjects manipulated to carry disease mutations or affected individuals carrying specific disease-causing mutations allowing for detailed evaluation of cellular phenotype, pharmacological manipulation, and/or toxicological screening. In this chapter, we will discuss HPSC differentiation into neural stem cells (NSCs) and neurons. We will review the mechanism underlying in vivo neural differentiation and methods which recapitulate this in vitro. We describe a method of deriving NSCs and differentiated mature neurons highlighting key steps of the core protocol. We also provide detailed information of the transcription factor and morphogen map of the developing brain which can be used as a guide to derive region- and lineage-specific NSCs and differentiated neurons. Key words Human pluripotent stem cells, In vitro neuronal differentiation, Directed differentiation, Induced differentiation, In vitro neuronal regionalization

1

Introduction

1.1 In Vivo Neural Development Mechanism

Neurodevelopment begins during gastrulation and can be divided into three main stages: (a) neural induction, the neuroectoderm undergoes morphological changes induced by “organizer tissue” to form the neural plate; (b) neurulation, the neural plate epithelial cells fold laterally to form the neural tube and neural crest; and (c) neural patterning, the neural tube differentiates into functionally and structurally distinct regions giving rise to the forebrain

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Fig. 1 Molecular mechanisms underlying in vivo neural induction, neurulation, and neural patterning (Modified and adapted from Zirra et al. 2016 [30]; Muguruma and Sasai 2012 [19]). (a) Neural induction—emergence of primary organizer tissue (AVE) alters the morphogen gradient promoting neural differentiation and assigns R-C and D-V axis to the developing neural tube. (b) Neurulation—neuroepithelial cells fold at its lateral ends forming the neural tube and neural crest cells. (c) Neural patterning—neural tube closes and differentiates into functionally and structurally distinct regions in a spatiotemporal manner giving rise to the characteristic forebrain (telencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon)

(telencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon) [1] (Fig. 1). The molecular mechanism underlying neural induction was first identified in Xenopus models with the discovery of the “organizer” tissue. By the mid-1990s, these “neural inducers” were

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identified as inhibitors of BMP signaling (chordin, noggin, and follistatin) [2, 3]. The neural tube comprises of rapidly dividing neuroepithelial cells uniquely responsible for the timely generation of neurons and glial cells in the developing brain [4]. The neural tube differentiates first along the R-C axis by caudalizing transcription factors from secondary organizer tissues (anterior neural ridge (ANR) and isthmic organizer (ISH)). These include FOXG1, OTX2, and SIX3 for forebrain development; FGF8, OTX2, and EN1 for the midbrain; FGF8, EN1, GBX2, and HOX for the hindbrain; and HOX for the spinal cord [5, 6] (Fig. 1). In addition, RA, FGF, and WNT secreted from the surrounding mesoderm and endoderm cells (caudal-high/rostral-low) complete the R-C regionalization. The D-V axis patterning commences thereafter by two opposing morphogen gradients produced by the roof plate secreting BMP4 and WNT and floor plate secreting SHH [7]. 1.2 In Vitro Recapitulation of Neural Induction

The potential of HPSCs as an unlimited source of patient and/or inaccessible tissue material begins with the quality of the starting material. HPSCs are routinely cultured as adherent cells in dishes with specific surface coating and media composition; thus, the combination of ECM and growth factors employed in a culture technique directly governs the success of downstream cell product [8]. Two main strategies are employed for in vitro neural induction: (a) directed differentiation and (b) induced differentiation. Directed differentiation involves in vitro mimicking of developmental signals by supplementing the culture media with morphogens, growth factors, and/or small molecules and is performed by either spontaneous differentiation (embryoid body) (Fig. 2b) or by using defined culture reagents targeting the mesodermectoderm signaling (dual SMAD inhibition) (Fig. 2b). Induced differentiation on the other hand involves neural induction without going through the process of pluripotency (Fig. 2c). This is achieved by direct conversion/transdifferentiation or direct reprogramming/dedifferentiation, wherein a somatic donor cell is converted to terminally differentiated induced neurons (iNS) or induced neural progenitors (iNPCs) by employing lineage-specific transcription factors (Fig. 2c).

1.3 Directed Differentiation

(a) Spontaneous (suspension induction)—This is performed by culturing HPSCs as single cell suspension to encourage cellcell interaction in basal media with complete withdrawal of all pluripotency-supporting factors via formation of multicellular 3D aggregates called “embryoid body” (EB) (Fig. 2b). Subsequent culturing in defined neural induction media (DMEM/F12, non-essential amino acid, heparin, and N2 supplement which provides insulin and transferrin) causes cavitation and formation of definitive neuroectoderm

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Fig. 2 Methods of neural induction and differentiation. (a) Schematic representation of different stages of neurodevelopment in vivo. (b) Directed differentiation—using cues from developmental stages and signaling pathways, two core protocols have been established, namely, the spontaneous/embryoid body method and the adherent monolayer dual SMAD inhibition method. Both methods result in generation of glia-like rosette neural epithelial cells characterized with trilineage differentiation potency. (c) Induced differentiation—two alternate strategies of generating patient-derived neuron are via direct conversion/transdifferentiation and direct reprogramming/dedifferentiation. Both methods employ overexpression of pioneer/master and lineagespecific transcription factor to alter the epigenetic signature of the donor somatic tissue making it amenable to desired fate conversion

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comprising of neuroepithelial cells (NPCs) giving rise to radially organized elongated cells called “neural rosettes” [9]. (b) Dual SMAD inhibition (monolayer induction)—This technique involves culturing HPSCs as adherent monolayers in defined culture (serum-free, feeder-free, and supplemented with N2) with BMP inhibition giving rise to rosette neuroepithelial cells (Rosettes-NPCs) (Fig. 2) [10]. While the media composition and pathway (BMP4/TGF-β/ Wnt) invoked are similar, the main difference between the above two protocols is the starting cell state, the duration of culture (4 weeks vs. 7–10 days), and the efficiency of conversion (monolayer higher efficiency). Several modifications to the EB protocol have been developed to overcome its limitations and lower efficiency [11]. The dual SMAD inhibition method provides a relatively easy to use technique and prolonged maintenance of potency in culture [12]. Thus, in vitro neural induction in HPSCs begins with the formation of “neural rosettes,” a polarized organization of neuroepithelial cells resembling the transverse section of the neural tube. These cells manifest all properties of the neural plate, including symmetrical cell division over prolonged periods in culture and expressing NPC markers like PAX6 and SOX1, Nestin, SOX1, SOX3, PSA-NCAM, and MUSASHI-1 [13]. These cells can be propagated either by dissociation (neurospheres) or adherent (monolayer) cultures in media containing growth factors (EGF, FGF, Wnt, Hedgehog, and TGF-β), ECM (laminin or Matrigel), and supplements (N2 and B27 which chiefly provides biotin, DL alpha tocopherol, vitamin A, BSA, catalase, insulin, transferrin, superoxide dismutase) [14]. NPCs so formed further differentiate to form radial glia (like) neural progenitor cells characterized with the requisite competence to respond to cell fate and positional cues provided in the culture media to differentiate into different neural cell types [14]. 1.4 Derivation of Neural Subtypes In Vitro Using Directed Differentiation

Cortical pyramidal neurons are predominantly projection glutamatergic neurons relaying information to distant areas of the brain. In basal differentiation media, NPCs show an inherent tendency to differentiate into telencephalic rostral (forebrain) and dorsal (cortical) resulting in mostly glutamatergic neurons [15]. Prior to terminal differentiation, NPCs if exposed to inhibitors of BMP, Wnt/β-ACTIN and TGF-β/ACTIVIN/NODAL (dual SMAD), and RA result in different layer-specific cortical neurons in a timespecific manner mimicking in vivo conditions [16]. Cortical GABAergic neurons comprise of both projection neurons (initiating from lateral ganglionic eminences (LGE) and

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inhibitory interneurons (initiating from medial ganglionic eminences (MGE). Prior to terminal differentiation, NPCs if exposed to ventralizing factors (inhibitors of SHH and/or Wnt) induce cortical GABAergic neuron [13]. Dopaminergic neurons arise from multiple regions of the brain with different functional specificities. Prior to terminal differentiation, NPCs if exposed to FGF8, SHH, and Wnt agonist induce caudal midbrain-specific dopaminergic neurons [17]. The cerebellar cortex is composed of molecular layer, Purkinje cell layer, and granule cell layer arising from two distinct tissues in the MHB (midbrain-hindbrain boundary)—the cerebellar ventricular zone producing Purkinje neurons and the rhombomere 1 producing granule cells [18]. Derivation of cerebellar neurons thus involves first respecification to caudal fate and followed by patterning along D-V axis. Prior to terminal differentiation, NPCs if exposed to FGF2 and insulin (weak caudalizing agents) at initial stages induce MHB regionalization followed by SHH inhibition (weak dorsalizing agent) which induces dorsal specification and results in Purkinje cells [19]. 1.5 Induced Differentiation

(a) Direct conversion/transdifferentiation—Transdifferentiation is achieved by directly inducing the epigenetic features of the desired cell type in the donor somatic tissue by a set of transcription factors (TFs) called “master” or “pioneer” TFs inducing cell fate change [20]. NGN2 is a human-specific master TF for neural conversion and in combination with NEUROD1, BRN2, and MYT1L converts human fibroblast into functional induced neurons (iNs) [21, 22]. NGN2 and BRN2 promote dorsal telencephalic cell fate giving rise to excitatory glutamatergic neurons [21, 23]. Co-overexpression of ASCL1 with striatum-enriched TFs (CTIP2, DLX1, DLX2) gives rise to functional GABAergic neurons [24], and co-overexpression of midbrain-specific TFs (LMX1A and NURR1) results in functional dopaminergic neurons [25]. (b) Direct reprogramming/dedifferentiation—This protocol involves altering the epigenetic signature of the somatic cells promoting fate transition. To generate induced neural progenitors (iNPCs), fibroblasts are first converted to an epigenetically unstable intermediate state, and then these cells are directed to neural fate by factors producing iNPCs with trilineage differentiation potential [26].

1.6 ThreeDimensional Neural Differentiations

Somatic cell-derived neurons from both directed and induced differentiation protocols meet functional characterization requirements. They fire action potential [12, 17], form functional synapse and region-specific projections when engrafted in host

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brain [12], express cell type-specific marker [27], and exhibit neuronal specification program [28]. However, there exist limitations and challenges to these protocols, like purity and efficiency of making specific neural subtype and inadequate mimicking of the cytoarchitectural complexity of the human brain impacting the informative content of the model. This has resulted in the establishment of in vitro 3D organoid (cell aggregate) recapitulating an organ’s development, structure, and function [28]. These 3D aggregates when cultured in suspension are characterized with a core comprising of radial glia-like NPCs, basal progenitors in the middle and differentiated neurons at the periphery [11]. When cultured within Matrigel droplets in spinning bioreactors, the 3D aggregates form cerebral organoid tissues characterized by an apicobasal membrane polarity and anterior-posterior axis resembling the developing human brain and can be maintained for a prolonged time in vitro [29]. The most significant observation of HPSC-derived neural induction and differentiation protocols as summarized above is the demonstration that several key components of cortical development occur through a high degree of self-organization of the derived NPCs both in 2D adherent and 3D suspension cultures. In the section below, we provide a detailed protocol for generating NSCs and differentiated cortical neurons using a combinatorial approach of embryoid body and dual SMAD inhibition to establish mature cortical neurons (Figs. 3 and 4). We further provide a detailed map of transcription factor and morphogen gradient (from established protocols) which can be used to direct the NSCs to produce region-specific NPCs and terminally differentiated neurons (Fig. 5).

2

Materials 1. Embryoid body media (EB): 20 mL KOSR, 1 mL Pen-Strep (100
stock) (Gibco-Life Tech, Cat #15140-122), 1 mL Glutamax (100
stock) (Gibco-Life Tech, Cat #35050-061), 1 mL NEAA (100
stock) (Gibco-Life Tech, Cat #11140050), and 180 μL beta-mercaptoethanol (Gibco-Life Tech, Cat #21985-023); make it up to 100 mL with KO DMEM (Gibco-Life Tech, Cat #10829-018). 2. Neural induction media (NIM): 1 mL N2 supplement (GibcoLife Tech, Cat #17502-048), 20 ng/mL bFGF (Peprotech Cat #100-18B-100 μg), 2 μg/mL heparin (Sigma, Cat #H3149), 1 mL Glutamax (100
stock) (Gibco-Life Tech, Cat #35050061), 1 mL NEAA (100
stock) (Gibco-Life Tech, Cat #11140-050), 1 mL Pen-Strep (100
stock) (Gibco-Life Tech, Cat #15140-122), 100 nM LDN-193189 (Sigma, Cat

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Fig. 3 In vitro recapitulation of neural induction and differentiation. (a) Derivation of neural stem cells using combinatorial approach of embryoid body and dual SMAD inhibition (SB431542, inhibitor of TGF-β, and LDN193189, inhibitor of BMP). (b) Differentiation of NSCs into mature cortical neurons

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Fig. 4 Cell state-specific characterization profile of HPSC-derived tissue material. (a) Top panel, immunostaining of pluripotency marker (OCT4 and SOX2); bottom panel, quantification of HPSC-specific markers indicating good-quality undifferentiated HPSC state (b) Top panel, immunostaining of NSC-specific markers (Nestin and SOX2); bottom panel, quantification of the NSC-specific markers indicating good-quality NSC state. (c) Top panel, mature neuronal marker MAP2 staining; bottom panel, whole cell recording membrane potential profile confirms neuronal functionality

#SML0559), and 10 μM SB-431542 (Sigma, Cat #S4317); make up to 100 mL with DMEM/F 12 (Gibco-Life Tech, Cat #10656-018). The medium is stable for 4 weeks when stored in the dark at 2  C to 8  C. To make larger volumes, increase the component amounts proportionally. 3. Neural expansion media (NEM): 1 mL N2 supplement (Gibco-Life Tech, Cat #17502-048), 2 mL B27 without vitamin A supplement (Gibco-Life Tech, Cat #12587-010), 20 ng/mL bFGF (Peprotech Cat #100-18B-100 μg), 2 μg/ mL heparin (Sigma, Cat #H3149), 1 mL Glutamax (100
stock) (Gibco-Life Tech, Cat #35050–061), 1 mL NEAA (100
stock) (Gibco-Life Tech, Cat #11140-050), and 1 mL Pen-Strep (100
stock) (Gibco-Life Tech, Cat #15140-122); make up to 100 mL with DMEM/F 12 (Gibco-Life Tech, Cat #10656-018). The medium is stable for 4 weeks when stored in the dark at 2  C to 8  C. To make larger volumes, increase the component amounts proportionally. 4. Neuronal differentiation media (NDM): 1 mL N2 supplement (Gibco-Life Tech, Cat #17502-048), 2 mL B27 with vitamin A supplement (Gibco-Life Tech, Cat #17504-044), 10 ng/mL

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Fig. 5 Map of transcription factor and morphogen gradient during neural development (Modified and adapted from Muruguma and Sasai 2012 [19]). Using a combination of small molecules and growth factor supplements in basal differentiation media allows for derivation of region-specific neural progenitors from the initially derived trilineage-competent neural stem cells. These progenitors respond to exogenous cues in media to enhance the specificity and efficiency of generating different neuronal subtypes

BDNF (Gibco-Life Tech, Cat #10908-010), 10 ng/mL GDNF (Gibco-Life Tech, Cat #PHC7045), 10 ng/mL IGF1 (Gibco-Life Tech, Cat #PHG0071), 1 mL Glutamax (GibcoLife Tech, Cat #35050-061), 1 mL NEAA (Gibco-Life Tech, Cat #11140-050), 1 mL Pen- Strep (Gibco-Life Tech, Cat #15140-122), 2 μM DAPT (5 mM stock) (Sigma Aldrich, Cat #D5942), and 20 ng/mL Activin A (1 μg/mL) (GibcoLife Tech, Cat #PHG9014); make up to 100 mL with DMEM/F 12 (Gibco-Life Tech, Cat #10656-018). The medium is stable for 4 weeks when stored in the dark at 2  C to 8  C. To make larger volumes, increase the component amounts proportionally. 5. Neuronal maintenance media (NMM): 1 mL N2 supplement (Gibco-Life Tech, Cat #17502-048), 2 mL B27 with vitamin A

HPSC Derived In-Vitro Neural Differentiation

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supplement (Gibco-Life Tech, Cat #17504-044), 10 ng/mL BDNF (Gibco-Life Tech, Cat #10908-010), 10 ng/mL GDNF (Gibco-Life Tech, Cat #PHC7045), 10 ng/mL IGF1 (Gibco-Life Tech, Cat #PHG0071), 1 mL Glutamax (GibcoLife Tech, Cat #35050-061), 1 mL NEAA (Gibco-Life Tech, Cat #11140-050), and 1 mL Pen-Strep (Gibco-Life Tech, Cat #15140-122); make up to 100 mL with DMEM/F 12 (GibcoLife Tech, Cat #10656-018). The medium is stable for 4 weeks when stored in the dark at 2  C to 8  C. To make larger volumes, increase the component amounts proportionally. 6. Other reagents: 50 μg/mL ascorbic acid (Sigma-Aldrich, Cat # A8960), 1
Dulbecco’s phosphate-buffered saline (Gibco-Life Tech, Cat # 14190-144), StemPro™ Accutase™ cell dissociation reagent (Gibco-Life Tech, Cat # A1110501), Matrigel (Corning Cat # 354277), 20 μg/mL poly-L-ornithine (Sigma, Cat. # P3655), 10 μg/mL laminin (Gibco-Life Tech Cat. #23017), 10 μM di-butyryl c-AMP (Sigma, Cat. #D0627), and distilled water (Gibco-Life Tech, Cat # 15230162). 7. Small molecule stock preparation: SB431542, stock concentration (dissolve in sterile DMSO) ¼ 10 mM, and LDN, stock concentration (dissolve in sterile distilled water) ¼ 1 mM. 8. Heparin stock preparation: Dissolve 25 mg of heparin sulfate in 1 mL of distilled water and mix until dissolved. After dissolving, filter through a 0.22 μm filter, aliquot 50–100 μL into sterile tubes, and store at 4  C. 9. bFGF stock preparation: Reconstitute bFGF with 5 mM Tris pH 7.54 with 0.1%BSA at a concentration of 0.1 μg/μL. Pre-chill the Eppendorf tubes and tips at 20  C. Add 500 μL of tris+BSA buffer into 50 μg vial of bFGF and mix. Aliquot 10 μL into sterile Eppendorf tubes under cold condition, and store at 20  C. (Note: Aliquoting to be done in 20  C cooler.) 10. Matrigel stock preparation: Aliquot Matrigel as per the dilution factor given in the certificate of analysis which can be downloaded by using lot number and catalogue number (the volume is typically between 270 and 350 μL). Store aliquots in 20  C. Add one aliquot in 25 mL of chilled DMEM/F12 and store at 4  C. (Note: Aliquoting to be done in 20  C cooler.) 11. Matrigel coating: Add 1.5 mL of Matrigel working solution to each 35 mm dish at least 1 h before plating the cells or overnight at 37  C. If using dishes with larger surface area, increase the volume accordingly.

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12. Plastic ware and equipment: Tissue culture-coated dishes, Falcons, cryovials, 0.22 μm filters, cell scrappers, and coverslip dishes. 13. Equipment: Laminar flow hood, inverted microscope, mechanical pipettor, and CO2 incubator.

3

Methods

3.1 Making Neural Stem Cells (NSCs) 3.1.1 Day 0: Generating Embryoid Bodies

1. Take a sub-confluent 35 colonies HiPSC culture previously maintained in standard HuES media supporting either feederdependent or feeder-free culturing (depending on the initial culture conditions). 2. Under a microscope, mark partially differentiating colonies or regions and manually scrape these using a sterile pipette tip. (Note: Use a dish that has more than 70% of the colonies in a compact undifferentiated state.) 3. Wash the dish with warm 1
PBS buffer. 4. Add an appropriate amount of Accutase™ cell dissociation buffer (Note: 200–300 μL for a 35 mm dish.) 5. Incubate the dish at room temperature or 37  C incubator for 2–3 min with gentle swirling. (Note: Incubation time may differ between cell lines, and therefore cell dissociation process should be standardized for each cell line.) 6. Gently triturate the cell suspension, and break the cell aggregates into single cell or small clumps using a cut 200 μL sterile pipette tip. 7. Transfer the cell suspension to a sterile 15 mL sterile falcon, and count the number of viable cells using trypan blue using a hemocytometer. 8. Centrifuge at 1200
g for 3 min at room temperature. 9. Carefully aspirate all the media and resuspend the pellet in sterile EB media (2.5 mL) supplemented with 10 μM SB-431542 and 100 nM LDN-193189 and dispense into a fresh non-adherent 35 mm culture dish. 10. Incubate the dish at 37  C.

3.1.2 Day 1–4: Partial Media Change

1. Warm (37  C) sufficient volume of EB media. 2. Gently remove about 1.5 mL of media, without disturbing the EBs and slowly add 1.5 mL fresh media supplemented with 10 μM SB-431542 and 100 nM LDN-193189.

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3. Incubate dish at 37  C and repeat steps 1–3 on alternate days till day 5. 3.1.3 Day 5–9: Neural Induction Media

1. Warm (37  C) sufficient volume of NIM. 2. Transfer the EBs from EB media to NIM media supplemented with 10 μM SB-431542 and 100 nM LDN-193189 in a non-adherent 35 mm dish. 3. Partial media change to be done as described above for 5 days.

3.1.4 Day 10: Plating EBs on Adherent Surface

1. Coat a 35 mm tissue culture dish with Matrigel at least an hour before plating cells. (Note: Matrigel coating can also be done at 37  C overnight and stored at 4  C for a week. Make sure that the ECM does not dry out.) 2. Warm (37  C) sufficient volume of NIM. 3. Collect the EBs using a large bore tip (or a cut 200 μL tip) so as to not break the EBs, and carefully transfer the suspension to a sterile 15 mL Falcon. 4. Let the EBs settle down in the tube and aspirate the spent medium leaving around 500 μL covering the EBs. 5. Gently remove the Matrigel solution from the new dish taking care that the coated surface is not scratched and dish not allowed drying out. 6. Add 2 mL of fresh NIM supplemented with 10 μM SB-431542 and 100 nM LDN-193189. 7. Gently transfer the EBs using large bore pipette (or a cut 200 μL tip) to the fresh dish and place the dish in the incubator. Move the dish in a circular motion so as to get all the EBs to the center. Leave undisturbed for 24 h.

3.1.5 Day 11–26: Rosette Formation and Enrichment

1. Warm (37  C) sufficient volume of neural induction media. 2. Aspirate carefully all the medium from the dish and replenish fresh medium supplemented with 10 μM SB-431542 and 100 nM LDN-193189. 3. Repeat steps 1 and 2 on alternate days until day 26 (or till we get enriched rosettes). 4. From days 13–14 onward, the EBs would have spread out, and neural rosettes should be visible. The neural rosettes will develop generally in clusters in most of the EBs. 5. The rosettes are isolated from the non-neural contaminating cells by cutting out the rosettes using a needle under sterile conditions using a dissection scope and passaged as above (steps 1 and 2) to get tertiary-enriched rosettes.

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(Note: Every fourth day, the rosettes are passaged if the enrichment is not complete till tertiary rosettes, and then it can be enriched to quaternary rosettes.) 3.1.6 Days 14–26: Neural Rosette Replating

1. Once rosettes are visible post plating of EBs, it is enriched by manual propagated till tertiary passage under the dissection scope. 2. The rosette clusters are pooled in a 15 mL Falcon tube and gently triturated with a wide bore tip (or 200 μL cut tip). 3. These rosettes can then be plated onto Matrigel pre-coated dishes in NEM. (Note: Small molecules SB-431542 and LDN-193189 can be used till tertiary rosette stage if there are many contaminating cells post primary rosette stage.)

3.1.7 Days 26–30: Neural Stem Cell (NSC) Propagation and Storage

1. The adherent NSCs can be passaged (split ratio of 1:2). To split the cells, use Accutase™ cell dissociation buffer as mentioned above (refer day 0 protocol). 2. The NSCs can be frozen in 9 FBS: 1 DMSO mixture or PSC cryomix.

3.2 Making Neurons from Neural Stem Cells (Pan-Neuronal Differentiation) 3.2.1

NSC Plating

1. Take a fully confluent NSC culture maintained in NEM. 2. Aspirate the spent media and discard it. 3. Wash the dish with warm 1
PBS and aspirate the buffer. 4. Add an appropriate amount of cell dissociation buffer Accutase™. (Note: 200–300 μL for a 35 mm dish.) 5. Incubate the dish at 37  C for 2–3 min. (Note: Incubation time may differ between cell lines, and therefore cell dissociation should be standardized for each cell line.) 6. Add warm 1 mL NEM to the dish and transfer the cell suspension to a sterile 15 mL Falcon. 7. Centrifuge at 300
g for 5 min at room temperature. 8. Carefully aspirate the supernatant without disturbing the pellet. 9. Resuspend the pellet in warm 1 mL sterile NEM. Count the number of viable cells using trypan blue using hemocytometer. 10. Calculate the number for cells needed (see Table 1) to initiate differentiation, and add the same in a sterile dish. (Note: The dish should be 70–80% confluent before starting neuronal induction.) 11. Incubate the dish for 12–16 h at 37  C.

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Table 1 NSC plating density for neuronal differentiation. Note: Pre-coat the destination dish with appropriate amount of Matrigel overnight or at least 1hour prior to cell plating Dish size

Approximate number of cells

Coverslip dish (14 mm glass diameter)

0.4
105 cells

12 well dish

2
105 cells

35 mm dish

6.5
105 cells

60 mm dish

13
105 cells

3.2.2 Neuronal Differentiation Day 0–10: Neuronal Induction

1. Pre-warm (37  C) sufficient amount of NDM. 2. Swirl the dish (to make sure all floaters are gathered at the center of the dish) and aspirate out the spent media from the dish. 3. Replace the media with warm 2 mL NDM. 4. Incubate the dish for 48 h at 37  C. 5. Repeat steps 1–4 every alternate day till day 10. (Note: To use these cells for multiple experiments, these can also be frozen at days 7–10 using 500 μL of PSC cryomix.)

Day 12–30: Neuronal Maintenance

1. Pre-warm (37  C) sufficient amount of NMM. 2. Slowly aspirate 1 mL media from the sides of the dish. (Note: Care should be taken to treat the dish gently henceforth so as to not dislodge the differentiating cells.) 3. Replace the media with warm 1 mL NMM. 4. Incubate the dish for 48 h at 37  C. 5. Repeat steps 1–4 every alternate day. Cells can be cultured in this condition for prolonged time (30–45 days).

References 1. Rakic P (2009) Evolution of the neocortex: a perspective from developmental biology. Nat Rev Neurosci 10(10):724–735 2. Sasai Y et al (1995) Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature 376 (6538):333–336 3. Fainsod A et al (1997) The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4. Mech Dev 63(1):39–50 4. Abranches E et al (2009) Neural differentiation of embryonic stem cells in vitro: a road map to neurogenesis in the embryo. PLoS One 4(7): e6286

5. Anderson RM et al (2002) Chordin and noggin promote organizing centers of forebrain development in the mouse. Development 129 (21):4975–4987 6. Wurst W, Bally-Cuif L (2001) Neural plate patterning: upstream and downstream of the isthmic organizer. Nat Rev Neurosci 2 (2):99–108 7. Hoch RV, Rubenstein JL, Pleasure S (2009) Genes and signaling events that establish regional patterning of the mammalian forebrain. Semin Cell Dev Biol 20(4):378–386 8. Efthymiou AG et al (2014) Self-renewal and cell lineage differentiation strategies in human

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embryonic stem cells and induced pluripotent stem cells. Expert Opin Biol Ther 14 (9):1333–1344 9. Zhang X et al (2010) Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell 7 (1):90–100 10. Kunath T et al (2007) FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from selfrenewal to lineage commitment. Development 134(16):2895–2902 11. Eiraku M et al (2008) Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3(5):519–532 12. Ho SM, Topol A, Brennand KJ (2015) From “directed differentiation” to “neuronal induction”: modeling neuropsychiatric disease. Biomark Insights 10(Suppl 1):31–41 13. Li XJ et al (2005) Specification of motoneurons from human embryonic stem cells. Nat Biotechnol 23(2):215–221 14. Dhara SK, Stice SL (2008) Neural differentiation of human embryonic stem cells. J Cell Biochem 105(3):633–640 15. Chambers SM et al (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27(3):275–280 16. Gaspard N, Vanderhaeghen P (2011) Laminar fate specification in the cerebral cortex. F1000 Biol Rep 3:6 17. Kriks S et al (2011) Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480 (7378):547–551 18. Hoshino M (2006) Molecular mechanisms underlying glutamatergic vs. GABAergic neuronal subtype specification in the cerebellum. Seikagaku 78(2):130–132 19. Muguruma K, Sasai Y (2012) In vitro recapitulation of neural development using embryonic

stem cells: from neurogenesis to histogenesis. Develop Growth Differ 54(3):349–357 20. Nizzardo M et al (2013) Direct reprogramming of adult somatic cells into other lineages: past evidence and future perspectives. Cell Transplant 22(6):921–944 21. Liu ML et al (2013) Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. Nat Commun 4:2183 22. Pang ZP et al (2011) Induction of human neuronal cells by defined transcription factors. Nature 476(7359):220–223 23. Heinrich C et al (2010) Directing astroglia from the cerebral cortex into subtype specific functional neurons. PLoS Biol 8(5):e1000373 24. Victor MB et al (2014) Generation of human striatal neurons by microRNA-dependent direct conversion of fibroblasts. Neuron 84 (2):311–323 25. Caiazzo M et al (2011) Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476 (7359):224–227 26. Ring KL et al (2012) Direct reprogramming of mouse and human fibroblasts into multipotent neural stem cells with a single factor. Cell Stem Cell 11(1):100–109 27. Maroof AM et al (2013) Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12(5):559–572 28. Suzuki IK, Vanderhaeghen P (2015) Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells. Development 142(18):3138–3150 29. Lancaster MA et al (2013) Cerebral organoids model human brain development and microcephaly. Nature 501(7467):373–379 30. Zirra A et al (2016) Neural Conversion and Patterning of Human Pluripotent Stem Cells: A Developmental Perspective. Stem Cells International 2016:1–14

Chapter 3 Generation of Definitive Neural Progenitor Cells from Human Pluripotent Stem Cells for Transplantation into Spinal Cord Injury Mohamad Khazaei, Christopher S. Ahuja, Christopher E. Rodgers, Priscilla Chan, and Michael G. Fehlings Abstract In this chapter, we first describe two interchangeable protocols optimized in our lab for deriving definitive neuronal progenitor cells from human pluripotent stem cells (hPSCs). The resultant NPCs can then be propagated and differentiated to produce differing proportions of neurons, oligodendrocytes, and astrocytes as required for in vitro cell culture studies or in vivo transplantation. Following these protocols, we explain the method for transplanting these cells into the rat model of spinal cord injury (SCI). Key words Neural progenitor cells, Pluripotent stem cells, Cell transplantation, Spinal cord injury

1

Introduction Neural progenitor cells (NPCs) are self-renewing, tripotent cells with the capacity to differentiate into neurons, oligodendrocytes, and astrocytes. NPCs are present during central nervous system (CNS) development and persist into adulthood in key locations. In the adult CNS, NPCs can be isolated from the subventricular zone (SVZ) of the forebrain, the subgranular zone (SGZ) of the dentate gyrus, as well as the central canal of the spinal cord [1–3]. While these cells are attractive for the treatment of neurological injuries due to their neural commitment and minimal risk of tumorigenicity [4–6], viable adult human NPCs are limited in supply, and ethical concerns surround the use of embryonic NPCs. Furthermore, allogenic grafts are likely to require immune modulation to avoid rejection. To bypass these challenges, it is possible to generate NPCs in vitro from pluripotent stem cells, including autogenic induced pluripotent stem cells (iPSCs), in near limitless quantities. As techniques have evolved, in vitro culture protocols have more closely mimicked in vivo developmental cues to consistently

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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produce desired cell populations. As an example, PSCs exposed to temporally controlled factors in vitro give rise to “rosette”-shaped structures, which can be identified via the expression of the transcription factors Pax6 and Sox2 and the intermediate protein Nestin [7]. This “neural rosette” resembles a developing neural tube and contains multipotent NPCs that have a similar expression profile as the neuroepithelial cells in the neural tube. As PSC-derived NPCs provide a scalable, self-renewing source of all three neural cell types, they are of critical significance for both neurodevelopmental studies and for the treatment of neurodegenerative diseases [8].

2 2.1

Materials Equipment

2.1.1 Procedure II: Generation of ClipCompression Spinal Cord Injury (SCI) in Rats

2.1.2

Procedure III

1. Rivlin-Tator modified Kerr-Lougheed ¼ curved aneurysm clip and applier (Fig. 2a, available from the Fehlings Laboratory). 2. Surgical instruments (Fine Science Tools Inc., Vancouver, BC, Canada): #15 scalpel, toothed forceps, fine sharp forceps, aim screw retractor, small self-retaining retractor, angled offset bone nippers, dull spinal hook, dissecting scissors, needle driver, staple applier, 3-O silk suture, and operating microscope. 1. Microinjector controller, microinjector, microinjection syringe (typically 1–10 μL). 2. Surgical instruments (as explained in Procedure II).

2.2

Reagents

2.2.1 Procedure I: Generation of Definitive Human NPCs Protocol 1

1. hPSC culture (hESCs or hiPSCs). 2. Fibroblast growth factors (FGFs; Wisent Inc., St-Bruno, QC, Canada). 3. Epidermal growth factor (EGF; Wisent Inc., St-Bruno, QC, Canada). 4. Heparin (sigma-Aldrich, St. Louis, MO, USA). 5. Neural expansion media (NEM). 6. Accutase® (Thermo Fisher Scientific, Waltham, MA, USA). 7. Matrigel (Corning, Corning, NY). 8. Notch ligand DLL4 (Peprotech, Rocky Hill, NJ, USA). 9. Poly-L-lysine (PLL) (Sigma-Aldrich, St. Louis, MO, USA). 10. Laminin (Thermo Fisher Scientific, Waltham, MA). 11. ROCK inhibitor (Y-27632; STEMCELL Technologies, Vancouver, BC, Canada).

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1. hPSC culture (hESCs or hiPSCs). 2. Fibroblast growth factors (FGFs; Wisent Inc., St-Bruno, QC, Canada). 3. Epidermal growth factor (EGF; Wisent Inc., St-Bruno, QC, Canada). 4. Heparin (Sigma-Aldrich, St. Louis, MO, USA). 5. Neural expansion media (NEM). 6. Neural induction media (NIM). 7. Accutase® (Thermo Fisher Scientific, Waltham, MA, USA). 8. Matrigel (Corning, Corning, NY, USA). 9. Notch ligand DLL4 (Peprotech, Rocky Hill, NJ, USA). 10. Poly-L-lysine (PLL) (Sigma-Aldrich, St. Louis, MO, USA). 11. Laminin (Thermo Fisher Scientific, Waltham, MA, USA). 12. ROCK inhibitor (Y-27632; STEMCELL Technologies, Vancouver, BC, Canada).

2.2.2 Procedure II: Generation of ClipCompression Spinal Cord Injury in Rats

1. Clavamox.

2.2.3 Procedure III: Transplantation of NPCs into Spinal Cord Injury

1. Cell suspension in an Eppendorf tube on ice.

2.3 Preparing Reagents

1. DMEM/F12 medium (Life Technologies, Carlsbad, CA, USA), supplemented with sodium pyruvate, Glutamax (Life Technologies, Carlsbad, CA, USA), 1% penicillin, streptomycin solution (Life Technologies, Carlsbad, CA, USA), N2 (Life Technologies, Carlsbad, CA, USA), B27 without vitamin a (Life Technologies, Carlsbad, CA, USA), 1% MEM (containing essential amino acids; Life Technologies, Carlsbad, CA), FGF2 (20 ng/mL), EGF (20 ng/mL), heparin (1.25 U/L; Scientific Protein Laboratories, Waunakee, WI, USA), TGFβ inhibitor (SB 431542; 10 μM; STEMCELL Technologies, Vancouver, BC, Canada), and BMP inhibitor (LDN 193189; 100 nM; STEMCELL Technologies, Vancouver, BC, Canada).

2.3.1 Neural Induction Medium (NIM)

2.3.2 NPC Expansion Medium (NEM)

2. Isoflurane.

1. DMEM/F12 medium (Life Technologies, Carlsbad, CA, USA) supplemented with sodium pyruvate, Glutamax (Life Technologies, Carlsbad, CA, USA), 1% penicillin, streptomycin solution (Life Technologies, Carlsbad, CA, USA), N2 (Life

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Technologies, Carlsbad, CA, USA), B27 without vitamin a (Life Technologies, Carlsbad, CA, USA), 1% MEM (containing essential amino acids; Life Technologies, Carlsbad, CA), FGF2 (20 ng/mL; Wisent Inc., St-Bruno, QC, Canada), EGF (20 ng/mL; Wisent Inc., St-Bruno, QC, Canada), and heparin (1.25 U/L; Scientific Protein Laboratories, Waunakee, WI, USA). 2.3.3

Matrigel Coating

1. Thaw a 5 mL vial of Matrigel (Corning, Corning, NY, USA) at 4  C overnight. Matrigel should form a gel-like texture at room temperature (or any temperature above 10  C). 2. The next day, add cold (~4  C) Matrigel to cold (~4  C) culture medium to a final concentration of 3 mg/mL. 3. Add 50 μL of diluted Matrigel to every 1 cm2 of growth surface, so as to cover the entire surface of each plate. 4. Warm up the plates for 1 h at 37  C, and then aspirate the remaining coating solution.

2.3.4 PLL/Laminin Coating

1. Make a poly-L-lysine (molecular weight of 30,000 to 70,000 Daltons; Sigma-Aldrich, St. Louis, MO, USA) solution of 0.1–1 mg/mL in 0.15 m borate buffer (pH 8.3; SigmaAldrich, St. Louis, MO), and then filter sterilize with a 0.2 μM filter. 2. Add enough solution to cover the surface of each plate, and then incubate at room temperature for 2 h. 3. Aspirate the solution, and then add the laminin solution (10 μg/mL) to the surface and incubate for 1 h at 37  C. 4. Aspirate the solution and wash gently with 1 PBS.

3

Methods

3.1 Procedure I: Generation of Definitive Human NPCs

Definitive human NPCs can be generated either with an embryoid body (EB) method or a dual-SMAD inhibition method (Fig. 1). To make NPCs which are fully committed to a neural lineage with full patterning and differentiation potential, we need to activate Notch signaling with a critically precise special and temporal dose. This treatment is important for the maintenance of a progenitor cell pool. Increasing the concentration or timing of Notch activation will result in the acquisition of glial identity cells [9]. A good culture of human pluripotent stem cells (hPSCs), sufficient in both cell quality and quantity, is needed for the generation of definitive NPCs. If hPSCs are cultured on a feeder layer, they should be acclimated to feeder-free conditions and be expanded in feeder-independent conditions for 3–4 passages before neural induction.

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Fig. 1 NPCs are generated from hPSCs based on the embryoid body or dual-SMAD inhibition method. The cells are going through the intermediate neural rosette stage and can be cultured and passaged using either the neurosphere or monolayer culture system 3.1.1 Procedure I.1: Generation of Definitive NPCs Using the Embryoid Body (EB)

The embryoid body formation technique is the older and more established method of NPC generation from hPSCs [7]. This method attempts to simulate the conditions of neurodevelopment to produce NPCs that more closely resemble the endogenous process and are thus potentially less likely to result in aberrant modifications (genetic/epigenetic/signaling, etc.) to resultant NPCs [7]. Considering the neuroectodermal lineage is the default pathway for pluripotent cell differentiation, the embryoid body formation technique is a generally simple, robust, and automatic procedure that requires few additional reagents beyond neuronal expansion media (NEM) for solely deriving Sox1+ neural rosettes and eventually nestin+, Sox2+, and Pax6+ NPC-containing neurospheres [7]. Although the EB method is generally lengthier (~40 days vs. ~23 for dual-SMAD inhibition), the overall yield of NPCs produced is generally higher [10]. Since the EB formation technique involves merely the crude selection of neural rosettes based solely on shape and position at the NPC differentiation phase, there is a risk, however small, for carrying over undesired pluripotent cells into the next stage of culture [8, 10]. Subsequently, NPCs will be generated by the sequential arrangement of EBs and neuroepithelial-like rosettes. The

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EB-derived rosettes can then be separated, gilded, and used as proliferative cells in the presence of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). Protocol

1. To begin neural induction, separate hPSCs to single cells with Accutase®, and then culture in suspension at the density of 1  105 cells/mL on low-adherent dishes in “neural expansion media (NEM)” (see Note 1). 2. The next day, replace half of the media with fresh media by tilting the plate. Such changes in half of the media should be made daily for a period of 5 days. From day 5 onward, cell aggregates, referred to as embryonic bodies (EB), should form. The three-dimensional structure of these EBs should generate a micro-tissue of embryonic structures, which acts to mimic the patterns observed within the naturally occurring transition from embryonic-origin pluripotent cells to neural tubelike cells. 3. On the fifth day of differentiation, EBs should be transferred to a 6 cm Matrigel-coated plate (Table 1) with NEM. After 24 h, carefully examine cells under the microscope. EBs should have settled and be fully adherent to the plate. 4. Change the media to fresh NEM on day 7. Change the media daily (using fresh NEM) until day 24. 5. On day 24, the neuroepithelial cells in the center of the colonies should form neural tubelike rosettes that are loosely attached. The first sign of the differentiation toward a neural lineage is the appearance of cells in the form of rosettes in the middle of the colonies, which occurs approximately 1 week after cultivation in NEM. The central, columnar cells in the

Table 1 Coating plates Matrigel coating Thaw a 5 mL vial of Matrigel at 4  C overnight. Matrigel should form a gel-like texture at room temperature (or any temperature above 10  C). The next day, add cold (~4  C) Matrigel to cold (~4  C) culture medium to a final concentration of 3 mg/mL. Add 50 μL of diluted Matrigel to every 1 cm2 of growth surface, so as to cover the entire surface of each plate. Warm up the plates for 1h at 37  C, then aspirate the remaining coating solution PLL/laminin coating

Make a poly-L-lysine (molecular weight of 30,000 to 70,000 Da) solution of 0.1–1 mg/mL in 0.15 m borate buffer (pH 8.3), then filter sterilize with a 0.2 μm filter. Add enough solution to cover the surface of each plate, then incubate at room temperature for 2 h. Aspirate the solution then add the laminin solution (10 μg/ mL) to the surface and incubate for 1 h at 37  C. Aspirate the solution and wash gently with 1 PBS

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rosettes, but not the cells in the periphery of each plate, will be positive for Pax6 (see Note 2). 6. After 2 days, manually pick up the rosettes using fine pipette tips. The outer, non-neuronal cells should remain on the plate. Note that NPC purity is strongly dependent on proper manual selection of rosettes. Collect neuronal rosettes in 15 mL falcon tubes in NEM (as many as necessary to accommodate the volume of rosettes), and then triturate/thresh the rosettes to separate individual cells. 7. The next step is to enrich NPCs by generating and isolating secondary rosettes. Resuspend the cells in NEM at a density of 1  105 cells/cm2 in the presence of Notch ligand DLL4 (500 ng/mL), and plate on PLL/laminin-coated plates (see Notes 3 and 4). At this stage, the resultant culture should now consist of pure populations of NPCs that are positive for the expression of Nestin, Pax6, and Sox2 (but not Oct4) [8, 11]. After this step, it is possible to culture cells with one of two culture techniques: (a) suspension neurospheres or (b) monolayer culture on substrate-coated plates. (a) Neurosphere System 8a. To culture cells using the neurosphere system, plate single cells at a clonal density of 10 cells/μL onto low-adherence culture dishes in NEM. Change half of the media every 2–3 days via tilting each dish. After 1 week, the cells should form primary neurospheres (containing primitive (still Oct4+) NPCs), approximately 50 μM in size (see Note 5). 9a. To passage the neurospheres, transfer the neurospheres into a 15 mL Falcon tube containing 500 μL NEM, and then triturate/thresh them by pipetting the media up and down (with a pasteur pipette) approximately 10 times each, or until they separate into single cells. Plate the individual cells at a clonal density of 10 cells/μm in a new dish. (b) Monolayer System 8b. To culture cells using a monolayer system, plate single cells at the seeding density of 1  10 4 cells/cm2 onto PLL/laminin-coated plates in NEM. Add ROCK inhibitor (10 μM) to the medium, and on the following day, change the media to NEM without ROCK inhibitor. 9b. After 5–6 days, the cells should have reached a sufficient confluency to be ready for passaging with Accutase® to new PLL/laminin-coated plates in NEM. Don’t forget to supplement the culture with ROCK inhibitor (10 μM) for the first day after passaging (see Note 6).

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Hereafter we will elaborate on the “dual-SMAD inhibition” method utilized by Chambers et al., with a chemically defined culture of adherent cells [12]. The fundamental principal behind this method is, as the name describes, the concept of inhibiting the action of the intracellular signaling proteins, SMADs, thus disrupting two cell signaling and differentiation pathways, (1) the bone morphogenetic protein (BMP) pathways and (2) the transforming growth factor beta (TGFβ) pathway, both of which act to inhibit the default pathway of neural induction [11, 12]. The dual-SMAD inhibition method presents the advantage of mitigating the risk of non-neural cells remaining alongside NPCs in culture, which is unavoidable to some degree with the EB formation technique [8, 12]. Additionally, dual-SMAD inhibition is generally faster than EB formation, taking approximately 21–23 days to produce a sufficient culture of usable/expandable definitive NPCs, in comparison to the EB technique, which can take up to 40 days to reach an equivalent point [8, 11, 12]. Despite these advantages, the final expected yield of NPCs produced will likely be lower with this method compared to the EB formation method [12].

3.1.2 Procedure I.2: Dual-SMAD Inhibition Method

1. To begin neural induction, separate hPSCs to single cells with Accutase®, and then culture the cells on Matrigel®-coated dishes at the seeding density of 25  104 cells/cm2 in “neural induction media (NIM)” (see Table 2). Supplement the media for initial seeding with 10 μM of Y-27632 (ROCK inhibitor) (see Note 7).

Protocol

2. Perform daily medium changes using fresh NIM supplemented with growth factors and morphogens, omitting the ROCK inhibitor (as it is no longer required after seeding cells). 3. In about 8–10 days after cultivation in NIM, the first signs of differentiation toward a neuronal lineage will be apparent by the formation of column-shaped cells in the form of rosettes situated in the middle of the colonies. At this stage remove all dual-SMAD inhibitors, and switch media to NEM (Table 2). Table 2 Culture media formulation Neural induction medium (NIM)

DMEM/F12 medium, supplemented with sodium pyruvate, glutamax, 1% penicillin, streptomycin solution, N2, B27 without vitamin A, 1% MEM (containing essential amino acids), FGF2 (20 ng/mL), EGF (20 ng/mL), heparin (1.25 U/L), TGFβ-inhibitor (SB 431542; 10 μM), BMP-inhibitor (LDN 193189; 100 nM)

NPC expansion medium DMEM/F12 medium supplemented with sodium pyruvate, Glutamax, 1% (NEM) penicillin, streptomycin solution, N2, B27 without vitamin A, 1% MEM (containing essential amino acids), FGF2 (20 ng/mL), EGF (20 ng/mL) and heparin (1.25 U/L)

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The next and final steps are similar to what is explained in steps 6–9 (in 3.1.1. Procedure I.1) for the EB method and are thus not repeated again here. 3.1.3 Inhibiting Notch Signaling

In both the EB and dual-SMAD inhibition procedures explained above, we explained the use of the Notch agonist delta-like 4 (DLL4) (500 ng/mL). While NPCS are actively proliferating, Notch signaling contributes to the maintenance of the undifferentiated state. Meanwhile, the resultant active self-renewing growth, in collaboration with FGF/EGF and Notch activation, increases the proportion of definitive NPCs [8]. Furthermore, the addition of DLL4 to NEM increases the self-renewal ability of NPCs—a method that has been established in our laboratory [8]. NPCs in the “neural rosette” state express the Notch signaling ligands JAG1, DLL1, and DLL4. The Notch1 receptor is enriched in the rosettes and mainly expressed at the inner circle of the rosette structures, facing the luminal side. However, when neural rosettes are dissociated to single cells during passaging, the level of Notch1 ligands drops, and at this time it is of critical importance to add Notch ligand and DLL4. A major advantage of using this method is that the resultant NPCs will be fully committed to a neural lineage identity. This allows the committed cells to be expanded and used for any in vitro culture experiments or in vivo transplantations where only neural cell types are desired [8, 11]. It is important to recognize that NPCs treated with DLL4 at this stage will undergo more progressive lineage specification toward terminally differentiated cell types as compared to cells which are not treated. Without the addition of DLL4 and Notch activation, NPCs produced from hPSCs may still express significant levels of some pluripotency genes (such as Oct4 and Nanog) [8]. It is however noteworthy that such aforementioned Notch activation can also direct the differentiation of NPCs toward a predominantly oligodendroglial fate (see below) and subsequently reduce the percentage of neurons generated. This oligodendroglial predisposition is of reduced concern if remyelination alone is the objective of any potential future transplantation of NPCs.

3.2 Procedure II: Generation of ClipCompression SCI in Rats

In our lab, we have developed a rodent clip compression model of SCI that closely mimics the human pathophysiology. The model allows investigation of injury mechanisms and testing of therapeutic interventions in a clinically relevant niche [13]. The model uses a modified aneurysm clip to create an initial contusive injury followed by a sustained compression, similar to the mechanism of most human injuries. Furthermore, this is a circumferential injury whereby the ventral, dorsal, and lateral spinal cord segments are affected allowing for the development of consistent

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intraparenchymal cystic cavitations over time as well as dense glial scarring. Other key features include titratable injury severity via adjustable clip strengths, reproducibility, and high fidelity by emulating the primary insult and secondary injury cascade occurring in humans [14–16]. While originally developed with immunocompetent rats [15], the model has also been translated to T-cell-deficient (Rowett nude; RNU) rats, which has facilitated the study of transplanted xenogenic (e.g., human, mouse) and allogenic cells (i.e., rat) without the need for daily immunosuppressive regimens [17]. Oral antibiotic prophylaxis (e.g., Clavamox in drinking water) is recommended for all animals beginning at least 2 days preoperatively and ending at sacrifice. The animals must remain deeply anesthetized from induction to recovery to limit movement during cord dissection and clip application. A mixture of nitrous oxide and isoflurane provides smooth induction and emergence. The anesthetic should be frequently adjusted based on the respiratory rate of the animal as SCI interferes with the animal’s ability to adequately ventilate. Standard operating procedures and humane animal care procedures should be strictly followed throughout the pre-, intra-, and postoperative course (Fig. 2).

Fig. 2 (a) Rivlin-Tator modified Kerr-Lougheed ¼ curved aneurysm clip. (b) Exposing the superficial and middle layers of the paraspinal muscles, (c) applying the clip, (d) closing the wound

hPSC-Derived Neural Progenitor Cells for SCI

35

1. Induce anesthesia using 5% inhaled isoflurane in a 1:1 N2O: O2 mix. 2. Quickly shave hair from the occiput to shoulder blades (T2), and measure the preoperative weight. 3. Adjust the nose cone, and utilize maintenance anesthesia of 2% isoflurane in 1:1 N2O:O2. Position the animal on a heating pad to maintain normothermia. Place a 1" stack of 2"  2" gauze pads under the neck and upper thorax of the animal to reduce cervical lordosis. 4. Apply masking tape over the eyes to prevent the animal’s corneas from drying out. Gently straighten the tail with tape to properly align the spine. 5. Inject the animal subcutaneously with saline, buprenorphine, and Metacam. Alternatively, injections may be done at the end of the procedure. 6. Apply two general skin preparations: (a) 70% ethanol twice (wait 3 min between preps) followed by (b) Betadine twice (wait 3 min between preps). Unpack sterile instruments, sterile towels, and don sterile gloves. Efficient exposure requires an understanding of important three-dimensional landmarks. The technique described here produces a C6–C7 injury; however, the laminectomy may be moved to C5–C6 or C7–T1 to produce, respectively, higher or lower injuries. 7. With a #15 blade and toothed forceps, make a midline incision through the skin and connective tissue of the occipital protuberance to the T2 prominence (the largest noticeable spinous process) to expose the paraspinal muscles. 8. Using small dissecting scissors with blades oriented mediolaterally, make a cut along the rostral face of the T2 spinous process to create a pocket beneath the superficial and middle layers of the paraspinal muscles. Orient the scissors dorsoventrally, and place the bottom blade inside this pocket to cut along the midline raphe (white line) from T2 to the occipital bone. 9. Apply one or two small self-retaining retractors to separate the muscles laterally. Retract the T2 spinous process caudally with a 3-0 silk suture to reduce cervical lordosis. 10. Use a #15 blade and fine forceps to split the deep paraspinal muscles along the midline to expose the spinous processes and laminae. 11. Retract the deep muscles until fully exposed. Scrape off all remaining muscles laterally with a scalpel up to the facet joints.

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12. Carefully cut through the ligamentum flavum along the caudal aspect of the C5, C6, and C7 laminae under which the dura mater should be exposed. 13. Use bone cutters to remove the C6 and C7 laminae bilaterally flush with the lateral masses. 14. Control bleeding with sterile/autoclaved cut gauze and/or Q-tips. The production of a consistent injury requires careful extradural dissection of the cord without infringing upon circulation or compressing the spinal cord. Even a short compression can cause damage to the delicate spinal cord. It is also imperative to avoid shearing or excessive retraction of the nerve roots as this will alter the injury phenotype. Finally, clip application can be difficult due to the expected epidural venous bleeding. This procedure is largely performed by tactile feedback and a thorough understanding of the anatomical structures ventral and lateral to the spinal cord. If CSF leakage occurs, this indicates an injury to the dura, and the procedure should be aborted. 15. Using a blunt spinal hook, carefully dissect the cord along the lateral aspects between the C6 and C7 nerve roots. 16. Beyond the curvature of the dura, use the spinal hooks to slowly dissect along the ventral aspect, extending all the way to the contralateral side. Epidural venous bleeding is expected and often cannot be controlled except by stopping the dissection. We recommend the completion of the dissection or a substantial portion of the dissection prior to clearing blood as it will re-accumulate on each attempt. 17. After the dissection is completed, with the hook in place, guide the open clip in the same way with the ventral blade over the hook. 18. Remove the spine hook. Quickly close the clip and start the timer. After 1 min, carefully open and remove the clip. Reduce isoflurane to 1% in 1:1 N2O:O2. Careful closure will facilitate skin and muscle healing and reduce infection/mortality among animals. Poor closure can also be a source of pain that can influence tests. 19. Irrigate the cavity with 5 mL of sterile saline. With the 3-0 silk or synthetic braided absorbable, suture the middle and superficial paraspinal muscle layers using a running or interrupted stitch. Suture the superficial cervical fascia in an analogous way such that no gaps are left. 20. Apply staples to the skin along the wound edges. Remove the adhesive strips and nose cone. Replace the animal in a warm cage in recovery position, alternating sides every 15 min until

hPSC-Derived Neural Progenitor Cells for SCI

37

awake. Place food pellets and drinking water on the floor of the cage. Bacon softies or kitten milk replacement may be provided as well. 3.3 Procedure III: Transplantation of NPCs into Spinal Cord Injury

Intraparenchymal transplantation of cells can be performed as required by the design of the study and may involve any cell type. The exact timing (acute, subacute, or chronic), side (unilateral, bilateral), region (e.g., perilesional), depth (gray matter, or white/superficial white matter), volume, and number of injections can be adjusted according to experimental requirements. Below we describe the general setup and a commonly used transplant approach. All transplant procedures involve (1) induction and preparation, (2) exposure, (3) transplantation, (4) closure, and (5) recovery (Fig. 3). Anesthetize the animal, and expose the C6/C7 level of the spinal cord as described above. For induction a lower dosage of anesthetic may be preferable as respiration is often compromised after injury. The exposure is similar to the injury procedure; however, significant scar tissue may obscure anatomical landmarks. In addition, the spinal cord no longer has a protective bony layer dorsally. We

Fig. 3 (a) Re-exposing the spinal cord for cell injection; (b) injecting the cells, (arrow points to the needle), (c) assembly of syringe for cell transplantation

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recommend performing the exposure of each stage further rostrally and caudally than in the original procedure to find the normal anatomy, and then dissect toward the previous operative site. After exposing the dura mater, cells can be transplanted. Intraparenchymal stem cell transplantation requires slow injections and gradual needle emergence to ensure that cells do not reflux from the needle tract. When inserting the needle, the entire bevel must be below the pia mater to ensure injection into the cord. When removing the needle, additional time may be required when reflux is seen. We use four injection sites, two sites 2 mm rostral and two sites 2 mm caudal to the lesion epicenter, with 50,000 cells/μL and a volume of 2 μL per site, which can be adjusted as per experimental requirements. 1. Position the injection apparatus over the first injection site. For our transplants, we choose injection sites that are 1–2 mm lateral to midline to avoid the rodent corticospinal tract. 2. Rinse the needle by aspirating and injecting 2 μL of normal saline. Load the required volume of cell suspension. 3. Under microscopic visualization, position the needle tip at the transplant site against the dura. Lift the needle tip, and puncture the dura/pia with a 30- or 32-gauge needle superficially to create a starting point for the transplant needle. 4. Gradually insert the tip of the needle into the spinal cord to the desired depth. The entire bevel must be deep in the pia mater. 5. Start the injection at a rate of 500 nL/min. Use caution to avoid reflux. 6. Wait for 2 min, and then slowly retract the needle by 1 mm. 7. Wait for another 2 min, and then fully pull back on the needle. If reflux is seen, carefully reinsert the needle. Wait for another 2 min. 8. Repeat steps 1–7 for each injection site. 9. To close the wound, gently irrigate the cavity with 5 mL of sterile saline. Do not apply water directly on the exposed cord. 10. Use 3-0 silk to suture the middle and superficial paraspinal muscle layers with a running or interrupted stitch. 11. Suture the superficial cervical fascia in an analogous way such that no gaps are created. 12. Apply staples to the skin along the wound edges. 3.3.1 Applications of NPCs for Treatment of Spinal Cord Injury

Traumatic spinal cord injury (SCI) is a devastating condition affecting as many as 40 out of every one million individuals in North America [18]. Traumatic SCI consists of an initial, primary physical injury causing direct damage to cells within the parenchyma followed by a secondary injury cascade of inflammation, ischemia, and

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39

cytotoxic molecule release. These injuries ultimately result in massive neuronal and oligodendroglial cell death through necrotic and apoptotic processes leading to large-scale demyelination and axonal disruption [18]. Although the glial scar has beneficial effects such as sequestering the spread of these pro-inflammatory signals and cell debris, the balance of evidence suggests it poses a challenge to successful regeneration and recovery in the chronic phase of injury. Over the long term, the scar acts as a physical and biochemical barrier to axonal regrowth and pro-regenerative cell migration, thus limiting the regenerative potential of endogenous or transplanted cells [18]. NPCs are one of the most promising cell types that have been studied thus far for the treatment of SCI due to their ability to replace lost neuronal circuits, remyelinate axons as oligodendrocytes, and provide local trophic support [19–21]. To date, NPCs and NPC-derived OPC grafts have been reported to significantly increase remyelination, improve axonal regeneration, and enhance recovery in preclinical models [4, 5]. Our lab and other labs have shown that the intraparenchymal transplantation of both rodent and human NPCs into the spinal cord can improve neural repair and functional recovery following traumatic SCI in rodents [6, 22–24]. The mechanisms of action of transplanted NPCs are incompletely understood but are likely multifactorial involving anatomic plasticity, remyelination, trophic support, neural circuit regeneration, and immunomodulation. Despite these exciting findings, human iPSC technology is only one decade old, and the full translational potential of hPSC-NPCs has not yet been realized. Nevertheless, hPSC-NPCs remain an exciting candidate for regenerative CNS therapy and will continue to grow as cell culture and transplant techniques evolve [17].

4

Notes 1. Starting with a homogeneous and healthy hPSC culture is important at this stage in order to achieve a higher efficiency of “pure” NPCs. 2. Neural rosettes at this stage are early neuroectodermal markers are capable of differentiating neuronal and glial cell types developmental cues.

comprised of cells expressing such as Pax6 and Sox1 and into various region-specific in response to appropriate

3. The density of cells for replating at this stage is critically important for determining the differentiation state of cells. After isolation of rosettes, they need to be replated in high density (1  105 cells/cm2) in the presence of Notch ligand DLL4 to maintain their rosette structure. In contrast, culturing at low

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plating densities results in increased levels of unwanted differentiation and a significant reduction in rosette formation efficiency. DLL4 treatment increases the rosette structures, expression levels of NPC marker genes, and proliferation potential of NPCs [25]. However, it should be noted that duration of DLL4 treatment is very important. Extended growth and passages at high cell densities and high DLL4 result in spontaneous differentiation and loss of rosette morphology. 4. Treatment with DLL4 should not be extended for more than one passage. Temporal activation of Notch signaling is important for the transition from primitive to fully definitive neural progenitor cell properties and for maintenance of the definitive state. At this stage transient activation of Notch signaling maintains stem cells in an uncommitted state and promotes their self-renewal. 5. Neurospheres should have a smooth shiny appearance and not have ragged edges and contain dark regions, vacuoles, nor dead cells. 6. By default, the hPSC-derived NPCs that are created using this method will have a dorsal anterior identity and be positive for the gene expression of orthodenticle homeobox 2 (OTX2), which encodes a homeodomain protein expressed in the foreand midbrain regions, yet be negative for the transcript for the homeobox protein, Hox-C4, an expression marker of spinal cord NPCs. 7. Starting with a homogeneous and healthy hPSC, culture is important at this stage in order to achieve a higher efficiency of “pure” NPCs. References 1. Weiss S, Dunne C, Hewson J et al (1996) Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 16:7599–7609 2. Gage FH (2000) Mammalian neural stem cells. Science 287:1433–1438 3. Emga˚rd M, Piao J, Aineskog H et al (2014) Neuroprotective effects of human spinal cordderived neural precursor cells after transplantation to the injured spinal cord. Exp Neurol 253:138–145 4. Keirstead HS, Nistor G, Bernal G et al (2005) Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci 25:4694–4705 5. Karimi-Abdolrezaee S, Eftekharpour E, Wang J et al (2006) Delayed transplantation of adult

neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury. J Neurosci 26:3377–3389 6. Cummings BJ, Uchida N, Tamaki SJ et al (2005) Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci U S A 102:14069–14074 7. Tao Y, Zhang S-C (2016) Neural subtype specification from human pluripotent stem cells. Cell Stem Cell 19:573–586 8. Salewski RP, Buttigieg J, Mitchell RA et al (2013) The generation of definitive neural stem cells from PiggyBac transposon-induced pluripotent stem cells can be enhanced by induction of the NOTCH signaling pathway. Stem Cells Dev 22:383–396

hPSC-Derived Neural Progenitor Cells for SCI 9. Gaiano N, Fishell G (2002) The role of notch in promoting glial and neural stem cell fates. Annu Rev Neurosci 25:471–490 10. Wen Y, Jin S (2014) Production of neural stem cells from human pluripotent stem cells. J Biotechnol 188:122–129 11. Smukler SR, Runciman SB, Xu S et al (2006) Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences. J Cell Biol 172:79–90 12. Chambers SM, Fasano CA, Papapetrou EP et al (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275–280 13. Ahuja CS, Fehlings M (2016) Concise review: bridging the gap: novel neuroregenerative and neuroprotective strategies in spinal cord injury. Stem Cells Transl Med 14. Ahuja CS, Martin AR, Fehlings M (2016) Recent advances in managing a spinal cord injury secondary to trauma. F1000Res 5 15. Forgione N, Karadimas SK, Foltz WD et al (2014) Bilateral contusion-compression model of incomplete traumatic cervical spinal cord injury. J Neurotrauma 31:1776–1788 16. Wilcox JT, Satkunendrarajah K, Nasirzadeh Y et al (2017) Generating level-dependent models of cervical and thoracic spinal cord injury: exploring the interplay of neuroanatomy, physiology, and function. Neurobiol Dis 105:194–212 17. Khazaei M, Ahuja CS, Fehlings MG (2017) Induced pluripotent stem cells for traumatic spinal cord injury. Front Cell Dev Biol 4 18. Ahuja CS, Wilson JR, Nori S et al (2017) Traumatic spinal cord injury. Nat Rev Dis Primer 3:17018

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19. Tsuji O, Miura K, Okada Y et al (2010) Therapeutic potential of appropriately evaluated safeinduced pluripotent stem cells for spinal cord injury. Proc Natl Acad Sci 107:12704–12709 20. Nori S, Okada Y, Yasuda A et al (2011) Grafted human-induced pluripotent stem-cell–derived neurospheres promote motor functional recovery after spinal cord injury in mice. Proc Natl Acad Sci 108:16825–16830 21. Kobayashi Y, Okada Y, Itakura G et al (2012) Pre-evaluated safe human iPSC-derived neural stem cells promote functional recovery after spinal cord injury in common marmoset without tumorigenicity. PLoS One 7:e52787 22. Karimi-Abdolrezaee S, Eftekharpour E, Wang J et al (2010) Synergistic effects of transplanted adult neural stem/progenitor cells, chondroitinase, and growth factors promote functional repair and plasticity of the chronically injured spinal cord. J Neurosci 30:1657–1676 23. Alexanian AR, Svendsen CN, Crowe MJ et al (2011) Transplantation of human glialrestricted neural precursors into injured spinal cord promotes functional and sensory recovery without causing allodynia. Cytotherapy 13:61–68 24. Emga˚rd M, Holmberg L, Samuelsson E-B et al (2009) Human neural precursor cells continue to proliferate and exhibit low cell death after transplantation to the injured rat spinal cord. Brain Res 1278:15–26 25. Woo S-M, Kim J, Han H-W et al (2009) Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells. BMC Neurosci 10:97

Chapter 4 Derivation of Neural Stem Cells from Human Parthenogenetic Stem Cells Rodolfo Gonzalez, Ibon Garitaonandia, Andrey Semechkin, and Russell Kern Abstract We have previously shown that human parthenogenetic stem cells (hpSC) can be chemically directed to differentiate into a homogeneous population of multipotent neural stem cells (hpNSC) that are scalable, cryopreservable, express all the appropriate neural markers, and can be further differentiated into functional dopaminergic neurons. Differentiation of hpSC into hpNSC provides a platform to study the molecular basis of human neural differentiation, to develop cell culture models of neural disease, and to provide neural stem cells for the treatment of neurodegenerative diseases. Additionally, the hpNSC that are generated could serve as a platform for drug discovery and the determination of pharmaceutical-induced neural toxicity. Here, we describe in detail the stepwise protocol that was developed in our laboratory that facilitates the highly efficient and reproducible differentiation of hpSC into hpNSC. Key words Parthenogenetic Transplantation

1

stem

cells,

Pluripotent,

Differentiation,

Neural

stem

cells,

Introduction Chemically activated unfertilized human oocytes develop into parthenogenetic blastocysts from which the inner cell mass can be isolated and expanded to generate stable hpSC cell lines [1]. hpSC are pluripotent stem cells that differentiate into derivatives of all three germ layers [1, 2]. hpSC have the same morphology, proliferation, and differentiation capacity as the biparental human embryonic stem cells (hESCs). hpSC can be heterozygous or homozygous depending on the way the genome forms from the maternal chromosome set. Homozygous hpSC can be derived from both heterozygous and homozygous donors, and their differentiated derivatives could potentially immune-match millions of patients if the HLA type is common [2, 3]. In addition to these immunogenic advantages, parthenogenesis does not destroy potentially viable human embryos, bypassing the ethical concerns

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_4, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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associated with hESCs. Therefore hpSCs represent an attractive pluripotent stem cell source for the derivation of clinically relevant somatic cell types, such as hpNSCs. hpNSCs are self-renewing multipotent stem cells of the central nervous system that differentiate into neurons, oligodendrocytes, and astrocytes [4]. hpNSC proliferate in vitro for several population doublings without losing their differentiation capacity and can be easily expanded into banks of billions of cells. After stringent quality control measures and extensive preclinical testing, these banks can be used for treating multiple neurological diseases such as Parkinson’s disease, traumatic brain injury, stroke, and spinal cord injury. We have previously reported a chemically defined feeder-free method for the derivation and expansion of hpNSC from hpSC [5]. This chemically directed differentiation method yields a highly pure population of hpNSC that can be expanded and cryopreserved in a robust and reproducible manner. Using a high-throughput screening strategy, we identified two potent neural inducers SB218078 and DMH-1 [5]. SB218078 is a structural homolog of stauprimide, which primes pluripotent hpSC for differentiation [6]. DMH-1 is a homolog of dorsomorphin, which efficiently induces neural conversion of pluripotent stem cells [7]. After 11 days of neural induction with these small molecules, the neuralized hpSC are dissociated and passaged 4–5 times to generate a pure population of multipotent hpNSC. Immunocytochemistry, flow cytometry, RT-PCR, gene expression microarray, and RNA sequencing confirmed the neural identity of the hpNSC and the lack of expression of pluripotency markers [5, 8]. The hpNSC maintain a normal karyotype and their proliferative and differentiation potential after cultivation, expansion, and cryopreservation, which significantly enhances their clinical application. These hpNSC are multipotent and differentiate into neurons, astrocytes, and oligodendrocytes and can be chemically directed to differentiate into pure population of dopaminergic neurons that express dopaminergic markers, secrete dopamine, and fire action potentials [5]. Additionally, our hpNSC have been shown to be safe and effective in treating Parkinson’s disease (PD) in animal models and have recently been granted clinical trial approval for the treatment of PD patients (ClinicalTrials.gov NCT02452723) [8–10]. hpNSC provide neurotrophic support, immunomodulation, and cell replacement to the damaged nigrostriatal system in PD. Here we describe a detailed step by step description of the derivation and expansion of hpNSC for clinical applications.

2

Materials All reagent preparation and cell culture handing should be carried out in a biological safety II cabinet under sterile conditions.

Human Parthenogenetic Stem Cell Derived Neural Stem Cells

2.1 General Equipment

45

1. Multi-gas incubator and CO2 and O2 control and humidified atmosphere. 2. Biological safety cabinet (biosafety level II). 3. Inverted bright-field microscope.

2.2 Chemicals and Growth Factors

1. Human FGF-basic recombinant (20 μg/mL) stock solution: Briefly centrifuge vial before opening and then reconstitute 10 μg by adding 0.5 mL of sterile 1 Dulbeccos’s phosphatebuffered saline (DPBS) without calcium and magnesium +0.1% BSA, and store in working aliquots at 20  C. Stock solution aliquots can be stored at 20  C for up to 6 months. 2. Human EGF recombinant (20 μg/mL) stock solution: Briefly centrifuge vial before opening and then reconstitute 10 μg by adding 0.5 mL sterile DPBS without calcium and magnesium +0.1% BSA, and mix and store in working aliquots at 20  C. stock solution aliquots can be stored at 20  C for up to 6 months. 3. SB218078 (10 mM) stock solution: To make a 10 mM stock solution, reconstitute 10 mg of SB218078 by adding 2.54 mL DMSO to entire contents of vial mix and store as 100 μL aliquots at 20  C. Stock solution aliquots can be stored at 20  C for up to 6 months. 4. DMH-1 (10 mM) stock solution: Reconstitute 10 mg by adding 2.63 mL of DMSO to entire contents of vial mix and store as 500 μL aliquots at 20  C. Stock solution aliquots can be stored at 20  C for up to 6 months. 5. Y-27632 ROCK inhibitor (10 mM) stock solution: Reconstitute 10 mg by adding 3.123 mL of DMSO to entire contents of vial, and mix and store as 500 μL aliquots at 20  C. Stock solution aliquots can be stored at 20  C for up to 6 months.

2.3 Culture Media and Solutions

1. Essential 8 Medium (Table 1): First thaw a frozen vial of Essential 8 Supplement overnight at 4  C. Aseptically add 10 mL Essential 8 Supplement to 490 mL of Essential 8 basal medium and store at 4  C (stable for 1 month). 2. Essential 8 Plating Medium: Aseptically add Y-27632 ROCK inhibitor to Essential 8 Medium, to a final 10 μM concentration. 3. Neural induction medium (Table 2): First thaw frozen N2 and B27 supplements overnight at 4  C. Aseptically add 5 mL of GlutaMax (100) solution, 5 mL N2 supplement (100) solution, and 10 mL B27 supplement (50) solution to 480 mL Knockout DMEM/F12 basal medium and store at 4  C (stable for 1 month). On the day of use, complete medium by adding SB218078 (to 5 μM final concentration) and DMH-1 (to 1 μM final concentration) using 10 mM stock solutions.

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Table 1 Essential 8 medium

Component

Company

Catalog #

Stock

Volume Final for 500 mL concentration Stored at

Essential 8 basal medium

Thermo Fisher Scientific

A10142-01

N/A

490 mL

N/A

A15171

50

10 mL

1

Essential 8 Thermo supplement Fisher Scientific

4 C

20  C

Table 2 Neural induction medium

Component

Company

Catalog #

Stock

Volume for 500 mL

SB218078

Tocris Bioscience

2560

10 mM

250 μL

5 μM

RT

DMH-1

Tocris Bioscience

4126

10 mM

50 μL

1 μM

4 C

Knockout DMEM/ F12

Thermo Fisher Scientific

12660-012

N/A

480 mL

N/A

4 C

GlutaMax-I

Thermo Fisher Scientific

35050-061

100

5 mL

1

4 C

N2 supplement

Life Technologies

17502-048

100

5 mL

1

20  C

B27 supplement

Life Technologies

0080085-SA 50

10 mL

1

20  C

Final concentration Stored at

4. StemPro NSC SFM medium (Table 3): First thaw a frozen vial of StemPro neural supplement overnight at 4  C. Aseptically add 5 mL GlutaMax (100) solution and 10 mL StemPro neural supplement to 484 mL Knockout DMEM/F12 basal medium. On the day of use, complete medium by adding 0.5 mL bFGF (20 μg/mL stock solution) and 0.5 mL EGF (20 μg/mL stock solution) from growth factor stock solutions. 5. CELLstart coating solution (1:50 diluted solution): To prepare 10 mL, add 0.2 mL CELLstart to 10 mL DPBS with calcium and magnesium (ThermoFisher Scientific, CA, USA) (see Note 1). 6. 1 phosphate-buffered saline (1 PBS): To prepare 1 L of 1 PBS, add 50 mL 20 PBS to 950 mL dH2O, mix, and adjust pH to 8.0 (see Note 2).

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Table 3 StemPro NSC SFM

Component

Company

Catalog #

Stock

Volume for 500 mL

Knockout DMEM/ F12

Thermo Fisher Scientific

12660–012

N/A

484 mL N/A

StemPro NSC Thermo SFM Fisher Scientific

A10508–01

50

10 mL

1

20  C

FGF basic

PeproTech

100-18B

20 μg/mL

500 μL

20 ng/mL

20  C

EGF

PeproTech

AF-100-15

20 μg/mL

500 μL

20 ng/mL

20oC

Glutamax-I

Thermo Fisher Scientific

35050–061

100

5 mL

1

Final concentration Stored at 4 C

4 C

7. Permeability solution (1 PBS/0.3% Triton X-100): To prepare 10 mL, add 30 μL Triton X-100. 8. Blocking solution (1  PBS/5% normal serum/0.3% Triton X-100): To prepare 10 mL, add 0.5 mL normal serum from the same species as the secondary antibody (e.g., normal goat serum to 9.5 mL 1 PBS) and 30 μL Triton X-100. 9. Wash buffer (1  PBS/0.1% Triton X-100): To prepare 500 mL, add 500 μL Triton X-100 to 500 mL 1 PBS and mix well. 10. Antibody dilution buffer (1 PBS/1% BSA/0.3% Triton X-100): To prepare 10 mL, add 30 μL Triton X-100 to 10 mL 1 PBS. Mix well and then add 0.1 g BSA; mix. 2.4 CELLstart Coated Vessels

1. Aseptically add an appropriate volume of CELLstart coating solution to the culture vessels, refer to table for recommended volumes (Table 4). Incubate at 37  C in a humidified atmosphere of 5% CO2 for at least 1–2 h (see Note 3). 2. Aspirate diluted CELLstart solution from the culture container and discard. Culture vessel is ready for plating cells.

3

Methods These methods describe the growth and differentiation of hpSC into hpNSC. Successful differentiation should result in 70–90% of cells expressing proteins that are characteristic of neural stem cells including Nestin, Musashi, and SOX2.

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Table 4 CELLstart recommended coating volumes Culture vessel Well of 24-well plate

Approximate surface area (cm2)

Volume of CELLstart solution needed

2

148 μL per well

2

1.9 cm

Well of 12-well plate

3.8 cm

27 μL per well

35 mm Petri dish

9 cm2

702 μL per dish

Well of 6-well plate

2

9.5 cm

741 μL per well

21 cm

2

1.638 mL per dish

T75 culture flasks

75 cm

2

5.850 mL per flask

T225 culture flasks

225 cm2

60 mm Petri dish

3.1 Thawing and Plating hpSC

17.550 mL per flask

1. Remove a vial containing 1  106 hpSC from the liquid nitrogen storage and immerse in a 37  C water bath without submerging the cap. Swirl the vial gently to promote thawing (see Note 4). 2. When only an ice crystal remains (1 min), remove the vial from the water bath, spray the outside of the vial with 70% ethanol to sterilize, and place it in hood. 3. Transfer the thawed hpSC cell suspension dropwise into a sterile 15 mL conical tube containing 10 mL of Essential 8 Plating Medium. 4. Rinse the vial with 1 mL of Essential 8 Plating Medium (containing 10 μM Y-27632 ROCK inhibitor) and then add it dropwise to the 15 mL conical tube with cells. 5. Centrifuge the cells at 200  g for 5 min at room temperature. 6. Aspirate and discard supernatant. 7. Resuspend the cell pellet with 5 mL Essential 8 Plating Medium (supplemented with 10 μM Y-27632 ROCK inhibitor) by gently pipetting up and down in the tube a few times (see Note 5). 8. Slowly add the thawed cell suspension onto 1 well of a 6-well plate pre-coated with CELLstart. Place the dish gently into a 37  C, 5% CO2 incubator and incubate the cells overnight. 9. The next day, aspirate the medium from the dish and replace it with pre-warmed Essential 8 Medium (without Y-27632 ROCK inhibitor). 10. Examine cells under the microscope and replace medium daily.

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Fig. 1 Phase-contrast images of hpSC cultures plated on CELLstart-coated plates at 1  105 cells/cm2 seeding density. The phase-contrast images show hPSC cultures at day 3 to day 5 after cell plating

Fig. 2 Phase-contrast images of hPSC cultures that are treated with Accutase 3.2

Passaging hpSC

1. hpSC cultures should be ready to be passaged once they reach ~85% confluency, approximately ~3–4 days after plating (Fig. 1). 2. To passage hpSC, aspirate culture medium and add 1 mL of pre-warmed StemPro Accutase to cover the bottom of 1 well of a 6-well plate. 3. Incubate at 37  C, 5% CO2, humidified atmosphere. After 1–2 min of incubation, observe for signs of hpSC monolayer detachment under an inverted microscope at 4 magnification (Fig. 2). 4. After 90% of the cells have detached (see Fig. 2), aseptically add an equal volume of Essential 8 Plating Medium (supplemented with 10 μM Y-27632, ROCK inhibitor). 5. Transfer the cell suspension into a 50 mL centrifuge tube and centrifuge at 200  g for 5 min.

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Table 5 Recommended volumes for growth and differentiation mediums Culture vessel type

Approximate surface area (cm2)

Essential 8 medium volume

1.9 cm

2

1 mL

Well of 12-well plate

3.8 cm

2

2 mL

35 mm Petri dish

9 cm2

Well of 24-well plate

Well of 6-well plate 60 mm Petri dish

4 mL

9.5 cm

2

5 mL

2

8 mL

2

21 cm

T75 culture flasks

75 cm

15 mL

T225 culture flasks

225 cm2

50 mL

6. Carefully aspirate the supernatant without disturbing the cell pellet. Resuspend the cell pellet with 30 mL of Essential 8 Plating Medium (supplemented with 10 μM Y-27632 ROCK inhibitor). 7. Transfer 5 mL of cell suspension to each well of a CELLstart pre-coated 6-well plate to achieve a 1:6 split ratio. 8. After 24 h of plating, replace medium with Essential 8 Medium (without Y-27632 ROCK inhibitor) to the culture vessels according to the Table 5. 9. Exchange medium every day until a cell monolayer covers at least 85% of the culture vessel growing surface. 10. The hpSC cultures should be ready for passage or differentiation once they reach 85% confluency, or once every 3–4 days. 3.3 Differentiation of hpSC into hpNSC

1. hpSC are ready for differentiation when they reach 85% confluency, about 3–4 days after plating at 1–2  105 cells/cm2 seeding density (Fig. 1). 2. Aspirate spent medium from hpSC cultures and wash cells once with 1 DPBS without calcium and magnesium. 3. Begin neural differentiation by adding neural induction medium (containing 5 μM SB218078 and 1 μM DMH-1) (see Table 5 for recommended volumes). Change medium every 2 days. 4. After 11 days of differentiation, aspirate Neural induction medium and wash cells hpNSC once with DPBS without calcium and magnesium. 5. Add room temperature Accutase to the culture vessel to cover the cell surface (see Table 6 for recommended volumes). Gently swirl the culture vessel to ensure complete coverage of the culture surface.

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Table 6 StemPro Accutase recommended volumes Culture vessel type

Approximate surface area (cm2)

Accutase volume

1.9 cm

2

200 μL

Well of 12-well plate

3.8 cm

2

300 μL

35 mm Petri dish

9 cm2

Well of 24-well plate

Well of 6-well plate

1 mL

9.5 cm

2

1 mL

21 cm

2

2.5 mL

T75 culture flasks

75 cm

2

8 mL

T225 culture flasks

225 cm2

60 mm Petri dish

25 mL

6. Incubate at 37  C in a humidified atmosphere of 5% CO2 in air, for 5 min. 7. After 90% of the cells have detached (see Fig. 1), aseptically add an equal volume of StemPro NSC SFM plating medium (supplemented with 10 μM Y-27632, ROCK inhibitor). 8. Pipette the cell suspension up and down several times to obtain single-cell suspension and then transfer to a 50 mL conical tube. 9. Perform cell count using a hemocytometer. 10. Centrifuge cell suspension at 210  g for 5 min. 11. Remove cell supernatant and resuspend cell pellet with StemPro NSC SFM plating medium (supplemented with 10 μM Y-27632). 12. Plate hpNSC on CELLstart-coated vessels at 1  105 cells/ cm2 seeding density. 13. After 24 h, remove spent medium and add fresh StemPro NSC SFM (without 10 μM Y-27632). 14. When the cells reach 90% confluence, passage hpNSCs at 1  105 cells/cm2 seeding density into CELLstart-coated tissue culture plates. 15. Passage 4–5 times to obtain a pure population of hpNSCs (see Note 6). 3.4 hpNSC Characterization

Successful differentiation of hpSC to hpNSC, as determined by immunohistochemistry or flow cytometry analysis, should result in 90% of the cells analyzed expressing proteins that are characteristic of neural stem cells including Nestin, Musashi, and SOX2 and not expressing pluripotency associated OCT-4 (Figs. 3 and 4).

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Fig. 3 Immunocytochemical analysis of hpNSC shows that the cells are positive for Nestin and Musashi and have the appropriate cell morphology

3.4.1 Immunohistochemistry

1. Using tweezers place sterile glass cover slips (12 mm diameter) into the bottom of wells of a 24-well plate. 2. Coat glass slides with CELLstart solution and then plate hpNSCs at 1  105 cells/cm2 seeding density. 3. When the cells reach 80–90% confluency (3–4 days after plating), aspirate StemPro NSC medium and then add 4% paraformaldehyde solution enough to cover the surface. 4. Allow hpNSCs to fix for 15 min at room temperature. 5. Aspirate fixative, rinse three times in 1 PBS for 5 min each. 6. Incubate fixed NSCs in permeabilization/blocking buffer for 60 min. 7. While blocking, dilute primary antibodies Nestin (Abcam, 1:100), Musashi (Abcam,1:300), and SOX2 (Abcam, 1:100) in antibody dilution buffer. 8. Incubate cells with primary antibody solution overnight at 4  C. 9. Rinse three times in 1 PBS solution for 5 min each. 10. Incubate cells in fluorochrome-conjugated secondary antibody diluted (1:500) in antibody dilution buffer for 1–2 h at room temperature in the dark. 11. Wash three times in 1 PBS for 5 min each. 12. Coverslip slides with Antifade reagent with DAPI.

Human Parthenogenetic Stem Cell Derived Neural Stem Cells

99.1%

Count

500

500

1,000

1,000

0.0%

102

103

104

105

106

107.2

101

103

104

105

106

107.2

Nestin 1,600

MUSASHI

98.8%

1,000

Count

1,000

96.7%

101

102

103

104

FL3-A

105

106

107.2

0

0

Count

102

FL1-A

2,000

3,200

FL3-A

500

101

0

0

Count

SOX2

1,600

1,600

OCT4

53

101

102

103

104

105

106

107.2

FL2-A

Fig. 4 Flow cytometry analysis of neural stem cell markers Musashi, Nestin, SOX2, and pluripotency marker OCT-4. Representative flow cytometry data shows that hpNSC were more than 90% positive for neural markers and negative for pluripotency markers. Percentage of positive cells (red) is calculated based on isotype control stained cells (blue)

3.4.2 Flow Cytometry Analysis

1. Harvest hpNSC with StemPro Accuatase and divide the cell suspension equally into four 15 mL conical tubes. 2. Centrifuge cells at 200  g for 5 min, aspirate, and discard supernatant. 3. Wash hpNSC by resuspending the cell pellets once with 10 mL 1 PBS. 4. Centrifuge hpNSC cell suspension at 200  g for 5 min and discard 1 PBS supernatant. 5. Fix hpNSC by resuspending cell pellets with 4% paraformaldehyde and incubating the cell suspension for 30 min at room temperature.

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6. Centrifuge hpNSC cell suspension at 200  g for 5 min and discard 4% paraformaldehyde supernatant. 7. Wash hpNSC twice by resuspending the cell pellets with 10 mL 1 PBS each time. 8. Centrifuge hpNSC cell suspension at 200  g for 5 min and discard 1 PBS supernatant. 9. Resuspend hpNSC cell pellet with permeabilization/blocking buffer and incubate cell suspension for 1 h at room temperature. 10. While blocking, dilute primary antibodies Nestin (Abcam, 1:20), Musashi (Abcam, 1:20), SOX (Abcam, 1:100), and OCT-4 (BD Biosciences, 1:20) in antibody dilution buffer. 11. After 1 h in permeabilization and blocking solution, centrifuge hpNSC cell suspension at 200  g for 5 min and discard supernatant. 12. Carefully resuspend cell pellets with primary antibodies solutions and incubate overnight at 4  C. 13. Wash hpNSC three times with 10 mL. Wash solution each time. 14. Centrifuge hpNSC cell suspension at 200  g for 5 min and discard Wash Solution supernatant. 15. Carefully resuspend cell pellets with secondary antibodies solutions and incubate 1 h at room temperature. 16. Centrifuge hpNSC cell suspension at 200  g for 5 min and discard antibody supernatant. 17. Wash hpNSC three times by resuspending the cell pellets with 10 mL Wash Solution each time. 18. Analyze on flow cytometer. 3.5

Freezing hpNSC

1. When hpNSC are 85–90% confluent, aspirate the StemPro NSC SFM medium from hpNSC cultures. 2. Wash the hpNSC once with DPBS without calcium and magnesium. 3. Add enough room temperature StemPro Accutase to cover the cell surface (see Table 6 for recommended volumes). Gently swirl the culture vessel to ensure complete coverage of the culture surface. 4. Incubate at 37  C in a humidified atmosphere of 5% CO2 in air, for 5 min. 5. After 90% of the cells have detached (see Fig. 1), aseptically add an equal volume of StemPro NSC SFM plating medium (supplemented with 10 μM Y-27632, ROCK inhibitor).

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6. Pipette the cell suspension up and down several times to obtain single cell suspension and then transfer to a 50 mL conical tube. 7. Perform cell count using hemocytometer. 8. Centrifuge the hpNSCs at 200  g for 5 min. 9. Remove cell supernatant and resuspend cell pellet with CryoStor CS10 freezing medium (BioLife Solutions, Bothell, WA, USA) to achieve 1–10  106 hpNSC/mL. 10. Transfer 1 mL of hpNSC suspension into pre-labeled, prechilled (4  C) cryovial tubes (see Note 7). 11. Transfer the cryovials to a Nalgene Mr. Frosty freezing container or controlled rate freezer and place container into a 80  C freezer. 12. With Mr. Frosty, transfer the cells the next day to liquid nitrogen. With controlled rate freezer, transfer the cells to liquid nitrogen after run. 3.6 Preparation of hpNSC for Transplantation

1. Check the confluence of the hpNSC culture under an inverted microscope. hpNSC are ready for transplantation when they reach 80–90% confluency (see Note 8). 2. Aspirate culture medium, and wash the cells once with DPBS without calcium and magnesium. 3. Add enough room temperature StemPro Accutase to cover the cell surface (see Table 6 for recommended volumes). 4. Incubate at 37  C in a humidified atmosphere of 5% CO2 in air, for 1 min. 5. After 90% of the hpNSCs have detached, aseptically add an equal volume of StemPro NSC SFM plating medium (supplemented with 10 μM Y-27632, ROCK inhibitor). 6. Pipette the cell suspension up and down several times to obtain single cell suspension. 7. Perform a cell count using a hemocytometer. 8. Centrifuge the hpNSC at 200  g for 5 min. Aspirate the supernatant and discard. 9. Resuspend pellet with DPBS to achieve 100,000 cells per μL (use cell count results from step 7 to determine volume of DPBS to add). 10. Transfer cell suspension to sterile microcentrifuge tube for the transfer to operating room.

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Notes 1. Do not freeze, and avoid vortexing and excessive agitation since this may cause gelling. 2. Prepare solutions with reverse osmosis deionized grade water (dH2O). 3. It is recommended to coat the culture vessels the day of use or the day before. 4. Do not use an incubator or the palm of your hand to thaw cell cultures since the rate of thawing achieved is too slow resulting in loss of viability. Use a water bath as described in the protocol above. 5. The addition of ROCK inhibitor to Essential 8 Medium will increase the viability of cells during plating. 6. It is very important to passage hpNSC 4–5 times to obtain a pure population of hpNSC without pluripotent stem cells. 7. The transfer of cell suspension to cryovials can be automated with a vial filler to minimize the time the cells are in contact with cryoprotectant. 8. Harvesting hpNSC at 100% confluency should be avoided as it may lead to decrease viability after transplantation.

References 1. Revazova ES, Turovets NA, Kochetkova OD, Kindarova LB, Kuzmichev LN, Janus JD, Pryzhkova MV (2007) Patient-specific stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 9(3):432–449. https://doi.org/10.1089/clo.2007.0033 2. Revazova ES, Turovets NA, Kochetkova OD, Agapova LS, Sebastian JL, Pryzhkova MV, Smolnikova VI, Kuzmichev LN, Janus JD (2008) HLA homozygous stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 10(1):11–24. https://doi.org/10.1089/clo.2007.0063 3. Taylor CJ, Bolton EM, Pocock S, Sharples LD, Pedersen RA, Bradley JA (2005) Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching. Lancet 366(9502):2019–2025. https://doi.org/10.1016/S0140-6736(05) 67813-0 4. Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255(5052):1707–1710

5. Gonzalez R, Garitaonandia I, Abramihina T, Wambua GK, Ostrowska A, Brock M, Noskov A, Boscolo FS, Craw JS, Laurent LC, Snyder EY, Semechkin RA (2013) Deriving dopaminergic neurons for clinical use. A practical approach. Sci Rep 3(1463):1–5. https:// doi.org/10.1038/srep01463 6. Zhu S, Wurdak H, Wang J, Lyssiotis CA, Peters EC, Cho CY, Wu X, Schultz PG (2009) A small molecule primes embryonic stem cells for differentiation. Cell Stem Cell 4(5):416–426. https://doi.org/10.1016/j.stem.2009.04.001 7. Zhou J, Su P, Li D, Tsang S, Duan E, Wang F (2010) High-efficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor beta superfamily receptors. Stem Cells 28 (10):1741–1750. https://doi.org/10.1002/ stem.504 8. Gonzalez R, Garitaonandia I, Crain A, Poustovoitov M, Abramihina T, Noskov A, Jiang C, Morey R, Laurent LC, Elsworth JD, Snyder EY, Redmond DE Jr, Semechkin R

Human Parthenogenetic Stem Cell Derived Neural Stem Cells (2015) Proof of concept studies exploring the safety and functional activity of human parthenogenetic-derived neural stem cells for the treatment of Parkinson’s disease. Cell Transplant 24(4):681–690. https://doi.org/ 10.3727/096368915X687769 9. Gonzalez R, Garitaonandia I, Poustovoitov M, Abramihina T, McEntire C, Culp B, Attwood J, Noskov A, Christiansen-Weber T, Khater M, Mora-Castilla S, To C, Crain A, Sherman G, Semechkin A, Laurent LC,

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Elsworth JD, Sladek J, Snyder EY, Redmond DE Jr, Kern RA (2016) Neural stem cells derived from human parthenogenetic stem cells engraft and promote recovery in a nonhuman primate model of Parkinson’s disease. Cell Transplant. https://doi.org/10.3727/ 096368916X691682 10. ClinicalTrials.gov (2016) A study to evaluate the safety of neural stem cells in patients with parkinson’s disease. https://clinicaltrials.gov/ ct2/show/NCT02452723

Chapter 5 Chemically Defined Neural Conversion of Human Pluripotent Stem Cells Yu Chen, Carlos A. Tristan, Sunil K. Mallanna, Pinar Ormanoglu, Steven Titus, Anton Simeonov, and Ilyas Singec¸ Abstract Human pluripotent stem cells (hPSCs) are characterized by their ability to self-renew and differentiate into any cell type of the human body. To fully utilize the potential of hPSCs for translational research and clinical applications, it is critical to develop rigorous cell differentiation protocols under feeder-free conditions that are efficient, reproducible, and scalable for high-throughput projects. Focusing on neural conversion of hPSCs, here we describe robust small molecule-based procedures that generate neural stem cells (NSCs) in less than a week under chemically defined conditions. These protocols can be used to dissect the mechanisms of neural lineage entry and to further develop systematic protocols that produce the cellular diversity of the central nervous system at industrial scale. Key words Pluripotency, Embryonic stem cell, Induced pluripotent stem cell, Neural induction, Cell differentiation, Culture medium, Coating substrate, Small molecules, Pathway inhibition

1

Introduction

1.1 Brief Overview: Neural Induction Strategies

Since the derivation of the first human embryonic stem cell (hESC) lines in 1998 by J. Thomson and colleagues [1], a number of protocols and their variations have been published aimed at differentiating pluripotent cells, including induced pluripotent stem cells (iPSCs) [2], into the derivatives of the three primary germ layers (ectoderm, mesoderm, endoderm). Early differentiation protocols mostly relied on embryoid body (EB) formation or overgrowth of cultures that lead to spontaneous and uncontrolled differentiation of pluripotent cells into mixed lineages [3, 4]. A strategy to enrich for neural cells takes advantage of the fact that some EBs, after plating on coated substrates such as laminin or Matrigel, can give rise to neural rosettes. These neural rosette structures, which mimic

Yu Chen and Carlos A. Tristan contributed equally to this work. Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_5, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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some aspects of the neural tube, can be manually collected under microscopic view or enzymatically detached from surrounding cell clumps and further expanded and differentiated into the three main neural lineages (neurons, astrocytes, oligodendrocytes) [4–6]. These protocols are difficult to standardize across laboratories, and it is apparent that different scientists employ varying preferences and practical routines when applying protocols. Investigator bias and uncontrolled strategies pose significant challenges for developing standard operating procedures (SOPs) for drug discovery and clinical applications. Co-culture of pluripotent cells with PA6 stromal cells exerting stromal-derived inducing activity (SDIA) was reported to enhance neural induction [7, 8]. However, co-culture of human ESCs with stromal cells up to 28 days was necessary to generate efficient numbers of PAX6-positive NSCs. The protracted neural induction process and the xenogeneic origin of PA6 cells are limiting factors for streamlined and efficient use of this approach for translational purposes. More recently, manipulation of specific cell signaling pathways that promote neural fate choice has emerged as a more controlled strategy. Consistent with knowledge accumulated on experimental model systems in developmental biology (e.g., Xenopus laevis), it was reported that BMP4 antagonizes recombinant Noggin (an inhibitor of BMP signaling) and promotes neural specification of human ESCs [7, 9, 10]. Importantly, activin/nodal signaling pathways contribute to pluripotency maintenance, and inhibition of these pathways promotes neural conversion of human cells [11–13]. More recently, inhibition of BMP, TGF beta, and WNT pathways was shown to direct pluripotent cells into the neural lineage although significant differences exist in protocols that use small molecule-based neural induction strategies [14, 15]. We therefore tested and compared various chemically defined conditions and small molecules that promote neural conversion of hPSCs (Figs. 1, 2, 3 and 4). 1.2 Media Formulations and Coating Substrates

Similar to mouse ESCs, the first hPSC lines were cultured in the presence of fetal bovine serum and irradiated mouse embryonic fibroblasts (MEFs) as supporting feeder layers [1]. The use of fibroblast growth factor 2 (FGF2) maintains pluripotency and supports the expansion of hESCs, which is different to mouse ESCs that rely on leukemia inhibitory factor (LIF). Over the last 20 years, the stem cell field has embarked on developing more chemically defined media, coating substrates, and alternatives to xenogeneic material as this would limit potential clinical use and regenerative medicine applications. For instance, reagents or procedures such as high concentrations of FGF2, knockout serum replacement (KSR), mTeSR, Matrigel, and laminin-521 are helpful for growing hPSCs under more defined conditions and in the absence of mouse feeders

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Fig. 1 Overview of neural induction strategies tested. Undifferentiated OCT4+ cell colonies are dissociated into single cells, counted, and plated onto dishes coated with VTN-N (50,000 or 200,000 cells/cm2). From days 0 to 5 medium, was changed daily using neural induction medium containing different combinations of small molecules. On day 6, PAX6 expression was analyzed using immunocytochemistry and FACS. Representative phase (PH) contrast images are shown on days 0, 3, and 6

[16–20]. As another important step forward, J. Thomson and colleagues developed an improved chemically defined medium that consists of eight components (DMEM/F12, L-ascorbic acid, selenium, transferrin, NaHCO3, insulin, FGF2, TGF beta-1). Of note, this culture system obviates the use of human serum albumin [21]. Using E8 medium allows successful and consistent growth of hESCs and iPSCs, and cells can be passaged using EDTA instead of enzymatic treatments, which may cause karyotype abnormalities and alter cell growth [22, 23]. Cells cultured in the presence of E8 medium are maintained on recombinant vitronectin (VTN-N), which is a truncated protein of human vitronectin corresponding to amino acids 62-478. Accordingly, for cell differentiation purposes E6 medium can be used, which is E8 medium devoid of pluripotency-promoting factors FGF2 and TGF beta-1. Based on a recent report, E6 medium alone is highly efficient in converting pluripotent cells into neural stem cells expressing PAX6+ [24], and we therefore performed comparison with DMEM/F12 medium (Figs. 2, 3, and 4). However, the results of this careful comparison and a recent report by Studer and colleagues [25] demonstrate the importance of inhibition of BMP and TGF beta pathways for most efficient neural induction. 1.3

Cell Density

Cell density and cell-cell contact are generally considered as important parameters for cell culture experiments using normal or cancerous cells (e.g., contact inhibition, proliferation rate, cell size,

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Fig. 2 Immunocytochemistry for PAX6 expression in hESCs (H9) cells after a 6-day neural induction. Comparison of E6 and DMEM/F12/N2B27 media and different combinations of small molecules. See Figs. 3 and 4 for quantification. Scale bars, 100 μm

mechanical force, metabolic adaptation). Indeed, growing PSCs at high cell densities can have various negative effects including DNA damage and genome instability [26]. Another paper [14] suggested that different cell densities may control hPSC differentiation into neuroectoderm (PAX6+) or neural crest (SOX10+), but more recently the same authors could not confirm a cell density effect on cell fate determination [25]. However, the neural induction protocols we tested here (low and high cell densities) indicate a clear cell density-dependent effect on neuroectoderm generation (Figs. 3 and 4). Therefore, we believe that controlling cell behavior based on providing appropriate culture conditions, including cell

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Fig. 3 Expression of PAX6 in hESCs (H9) detected by flow cytometry in DMEM/F12/N2B27 culture media using low (a) and high (b) cell densities (50,000 or 200,000 cells/cm2). After culturing hESCs in each culture

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density, is of importance for formulating reproducible cell differentiation protocols and developing robust and scalable assays for translational research.

2

Materials Human embryonic stem cell line WA09 (H9 ESC line, WiCell, Madison, WI, USA); results were reproduced with an iPSC line (iPSC-GR1.1, Lonza, Walkersville, MD; cell line generated on behalf of NIH Common Fund) 1. Essential 8 (E8) no. A1516401).

medium

(Life

Technologies

cat.

2. Essential 6 (E6) no. A1517001).

medium

(Life

Technologies

cat.

3. DMEM/F12 medium (Life Technologies cat. no. 10565018). 4. N2 supplement (Life Technologies cat. no. A1370701). 5. B27 supplement (Life Technologies cat. no. 12587010). 6. Accutase (Life Technologies cat. no. A1110501). 7. Vitronectin-N (VTN-N) (Life Technologies, cat no. A14700). 8. Dorsomorphin (Tocris, cat. no. 3093). 9. LDN-193189 no. SML0559).

hydrochloride

(Sigma-Aldrich,

cat.

10. A83-01 (Tocris, cat. no. 2939). 11. PNU-74654 (Tocris, cat. no. 3534). 12. ROCK inhibitor Y-27632 dihydrochloride (Tocris, cat. no. 1254). 13. Dulbecco’s phosphate-buffered saline (DPBS; Life Technologies, cat. no. 14040133). 14. DPBS without calcium or magnesium (Life Technologies, cat. no. 14190250). 15. UltraPure 0.5M no. 15575020).

EDTA

(Life

Technologies,

cat.

16. 6-well and 24-well tissue culture plates (Corning). 17. For immunocytochemistry rabbit anti-PAX6 antibody (Biolegend, cat. no. 901301). ä Fig. 3 (continued) condition for 6 days, cells were dissociated and analyzed for PAX6 expression with an antiPAX6 Alexa 488-conjugated antibody. Table shows percentage of PAX6+ cells in each culture condition tested. Dot plots represent PAX6 labeling for each culture condition, isotype and unstained controls. Histograms represent PAX6 labeling for each culture condition, isotype and unstained controls

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Fig. 4 Expression of PAX6 in hESCs (H9) detected by flow cytometry in E6 culture media using low (a) and high (b) cell densities (50,000 or 200,000 cells/cm2). After culturing hPSCs cells in each culture condition for

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18. For immunocytochemistry donkey anti-rabbit secondary antibody Alexa 488 (Thermo Fisher Scientific, cat. no. A-21206). 19. For flow cytometry anti-PAX6 Alexa Fluor 488-conjugated antibody (BD Pharmingen, cat. no. 561664). 20. For flow cytometry Alexa Fluor 488 isotype control (BD Pharmingen, cat. no. 565362). 21. Paraformaldehyde (PFA, Electron Microscopy Sciences, cat. no. 15714-S). 22. Bovine serum albumin (BSA; Cell Signaling Technology, cat. no. 9998S). 23. Fetal bovine serum (FBS; Sigma-Aldrich, cat. no. F4135). 24. Triton X-100 (Sigma-Aldrich, cat. no. T9284). 25. Hoechst (Life Technologies, cat. no. H3570). 26. Tween 20 (Affymetrix, cat. no. 20605). 27. Trypan blue (Life Technologies, cat. no. 15250061).

3 3.1

Methods Equipment

1. Incubator Cells are maintained at 37  C, 5% CO2 in Forma Steri-Cult CO2 incubators (Thermo Fisher Scientific, model #3310) outfitted with HEPA air filtration. 2. Microscopy Fluorescence images were taken using LEICA DMi8 epi-fluorescence microscopy equipped with HAMAMATSU CMOS camera ORCA-Flash4.0LT, DAPI and FITC filter sets. Settings for camera exposure time and excitation intensity are kept identical for images taken from different samples. Fluorescence images from the same channels are also presented using identical contrast settings. 3. Fluorescence-activated cell sorting (FACS) Cells were analyzed using a BD LSRFortessa flow cytometer (Model #647794L6) equipped with 405 nm, 488 nm, 561 nm, and 635 nm lasers.

ä Fig. 4 (continued) 6 days, cells were detached, dissociated, and analyzed for PAX6 expression with an antiPAX6 Alexa 488-conjugated antibody. Table shows percentage of PAX6+ cells in each culture condition tested. Dot plots represent PAX6 labeling for each culture condition, isotype and unstained controls. Histograms represent PAX6 labeling for each culture condition, isotype and unstained controls

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67

Sample acquisition was done using FACSDiva v6.1.3, and the data were analyzed with FlowJo v10 software. 4. Cell counter Cells were diluted 1:1 in trypan blue and counted using a Countess II FL automated cell counter (cat. no. AMQAF1000). 3.2 Propagation of Pluripotent Cells

1. Human ESCs and iPSCs are maintained under feeder-free and xeno-free condition using E8 medium and VTN-N following the instructions of the manufacturer. 2. Coat culture vessels with VTN-N (1:100; diluted in DPBS without calcium and magnesium). Use 1 mL of diluted VTN-N to coat one well of a 6-well, and incubate for 1 h at room temperature or overnight at 4  C sealed with parafilm. Aspirate VTN-N immediately before plating H9 cells, and make sure that plates don’t dry out. 3. H9 cells are passaged when grown to 70–90% confluence, typically every 4 to 5 days. 4. Passage cells using 0.5 mM EDTA diluted in DPBS (without calcium or magnesium). Remove E8 medium, wash once with DPBS, and then incubate with 0.5 mM EDTA for 5 min at 37  C. When H9 cells start rounding up, remove EDTA and add E8 medium. Pipet up and down gently to dissociate pluripotent cells into small clumps. Plate cells onto fresh plates at a splitting ratio of 1:6 to 1:12. 5. To promote cell recovery after dissociation, ROCK inhibitor Y-27632 (10 μM) can be used for 24 h but is typically not necessary for standard long-term maintenance of pluripotent cells if sufficient amount of cellular material is available at every passage.

3.3

Neural Induction

1. When grown to 70–90% confluence, wash pluripotent cells with DPBS, and then treat with Accutase for 5 min at 37  C. Use 1 mL Accutase for each well of a 6-well plate. 2. When cells start detaching, add 1 mL E8 medium to dilute Accutase. Gently detach cells from the culture dish by pipetting up and down. 3. Transfer cells into 15 mL conical tube, and centrifuge at 300  g for 5 min. 4. Gently remove media by aspiration (see Note 1). 5. Resuspend cell pellet in E8 medium and perform cell count. 6. Add ROCK inhibitor Y-27632 (10 μM) to increase cell viability.

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7. Remove VTN-N from the coated plates and plate cells at a density of 50,000 or 200,000 cells/cm2. 8. Twenty-four hours after cell plating, neural induction is initiated by switching from E8 medium to the various neural induction media, including DMEM/F12 + N2B27 or E6 medium supplemented with DA, DAP, LA, or LAP (see Note 2 and Subheading 3.4 for abbreviations). 9. Feed cells everyday with fresh neural induction media for 6 days. 10. In the presence of small molecular inhibitors, at day 6 this neural induction protocol will have produced more than 90% PAX6+ neural stem cells (see Note 3). 11. On day 6 cells can be analyzed for neural marker expression such as PAX6 expression by immunocytochemistry and flow cytometry (see Note 4 and Subheadings 3.5 and 3.6). 3.4 Comparison of Neural Induction Conditions

To allow direct comparison, the following base media and conditions were tested (Fig. 1): 1. E6 + 2 μM dorsomorphin + 2 μM A83-01 + 2 μM PNU-74654 (DAP) 2. E6 + 2 μM dorsomorphin + 2 μM A83-01 (DA) 3. E6 + 100 nM LDN-193189 + 2 μM A83-01 + 2 μM PNU74654 (LAP) 4. E6 + 100 nM LDN-193189 + 2 μM A83-01 (LA) 5. DMEM/F12 + N2B27 + DAP 6. DMEM/F12 + N2B27 + DA 7. DMEM/F12 + N2B27 LAP 8. DMEM/F12 + N2B27 + LA

3.5 Immunocytochemistry

The volume of reagents listed below applies for one well of a 24-well plate: 1. Wash cells 2 with 1 mL DPBS. Carefully add DPBS against the side of the well to avoid detaching the cells from the well. 2. Add 500 μL 4% PFA and fix for 30 min at room temperature. 3. Wash 3 with 1 mL DPBS. 4. Add 0.5 mL permeabilization/blocking buffer (DPBS, 0.3% Triton X-100, 5% BSA), and incubate for 1 h at room temperature. 5. Add rabbit anti-PAX6 antibody (1:200) diluted in Permeabilization/Blocking buffer, and incubate for 1 h at room temperature or overnight at 4 oC. 6. Wash 3 with 1 mL PBS.

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7. Add Alexa 488-conjugated donkey anti-rabbit secondary antibody (1:1000) diluted in permeabilization/blocking buffer, and incubate for 1 h at room temperature. 8. Wash 3 with 1 mL PBS. 9. Add Hoechst (1:4000) diluted in DPBS, and incubate for 30 min at room temperature. 10. Wash with DPBS, and perform microscopic analysis (see Note 5 and Fig. 2). 3.6

FACS

1. For flow cytometry remove culture media, rinse cells with DPBS (without calcium and magnesium), add Accutase (1 mL to each well of a 6-well plate), and incubate for 3–5 min to achieve singlecell dissociation. Wash cells off the well with 1 mL DPBS (without calcium and magnesium). Collect cells in a 15 mL conical tube, and centrifuge at 300  g for 5 min. Gently remove supernatant. Wash cells two times with DPBS (without calcium and magnesium) at 4  C. Centrifuge at 300  g for 5 min. 2. Resuspend cell pellet in 875 μL DPBS (without calcium and magnesium) at 4  C. Vortex to obtain a homogenous cell suspension. 3. Dropwise add 125 μL of 32% PFA to cell suspension while vortexing (final PFA concentration is 4%). Incubate at room temperature in fixation buffer for 30 min. Centrifuge at 300  g for 5 min. Wash cells three times with DPBS (without calcium and magnesium) at 4  C. 4. Resuspend cell pellet in 2 mL DPBS (without calcium or magnesium). Count cells and transfer 2  106 cells into a new 15 mL conical tube, centrifuge at 300  g for 5 min, and gently aspirate supernatant. 5. Resuspend cell pellet in permeabilization buffer (0.2% Tween 20 in DPBS), and incubate at room temperature for 20 min. Centrifuge at 300  g for 5 min, gently aspirate buffer and wash one time with DPBS. 6. Resuspend cell pellet in 1 mL of blocking buffer (0.5% BSA and 2% FBS in DPBS), and incubate on ice for 30 min. Centrifuge at 300  g for 5 min, and carefully aspirate supernatant. 7. Resuspend cell pellet in 200 μL of sorting buffer (0.5% BSA in DPBS) containing 0.05 μg of anti-PAX6 Alexa Fluor 488-conjugated antibody or 0.05 μg of Alexa Fluor 488 isotype control. Incubate on ice for 30 min in the dark. Centrifuge at 300  g for 5 min, and gently remove supernatant (see Note 6).

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8. Wash cells three times with permeabilization buffer while protecting cells from light to minimize photobleaching. Centrifuge at 300  g for 5 min, and gently aspirate supernatant. 9. Resuspend cell pellet in 500 μL of sorting buffer, and filter cells through a 40 μm cell strainer to remove cell clumps. 10. Analyze stained isotype and unstained control cells by flow cytometry (see Note 7 and Figs. 3 and 4)

4

Notes 1. To get accurate numbers, cell densities can be calculated using ImageJ or IncuCyte (Sartorius). 2. Use swing centrifuge with swing bucket rotor for pelleting cells, and resuspend cell pellet in a minimum of 1 mL. Using a fixed-angle centrifuge will result in a higher number of cell loss throughout protocol. 3. Neural stem cells at this stage are highly proliferative and can be further expanded using mitogens such as FGF2 and EGF (epidermal growth factor) or patterned to specific progenitors using morphogens such as retinoic acid (data not shown). 4. Cells can be plated onto 24-well plates depending on experimental design or to reduce the amount of antibody required for immunocytochemistry. 5. For immunofluorescence analysis, it is critical to include a negative control to determine level of background staining. Undifferentiated H9 cells grown in E8 medium for 6 days were used as a negative control for PAX6 staining. Once the level of background staining is established, the same threshold is applied to all the samples to subtract nonspecific staining from the acquired images. This is particularly important for automated imaging and high-content imaging. 6. If using non-conjugated primary antibodies, a second incubation with a fluorescent dye conjugated secondary diluted in sorting buffer is required. Always use isotype control at the same concentration (μg/μL) as the primary antibody. 7. For flow cytometry analysis, use software to set gates to subtract background from unstained cells and isotype control.

Acknowledgment We thank all our colleagues at the NIH National Center for Advancing Translational Sciences (NCATS) for their collaboration and the NIH Common Fund (Regenerative Medicine Program) for funding the Stem Cell Translation Laboratory (SCTL).

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References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocyst. Science 282:1145–1147 2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872 3. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T (2001) Neural progenitors from human embryonic stem cells. Nat Biotechnol 19:1134–1140 4. Zhang SC, Wernig M, Duncan ID, Bru¨stle O, Thomson JA (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19:1129–1133 5. Li XJ, Du ZW, Zarnowska ED, Pankratz M, Hansen LO, Pearce RA, Zhang SC (2005) Specification of motoneurons from human embryonic stem cells. Nat Biotechnol 23:215–221 6. Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, Studer L (2008) Human ES cellderived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev 22:152–165 7. Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa SI, Sasai Y (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cellderived inducing activity. Neuron 28:31–40 8. Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA 101:12543–12548 9. Pera MF, Andrade J, Houssami S, Reubinoff B, Trounson A, Stanley EG, Ward-van Oostwaard D, Mummery C (2004) Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J Cell Sci 117:1269–1280 10. Sonntag KC, Pruszak J, Yoshizaki T, van Arensbergen J, Sanchez-Pernaute R, Isacson O (2007) Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin. Stem Cells 25:411–418 11. Vallier L, Alexander M, Pedersen RA (2005) Activin/Nodal and FGF pathways cooperate to

maintain pluripotency of human embryonic stem cells. J Cell Sci 118:4495–4509 12. Smith JR, Vallier L, Lupo G, Alexander M, Harris WA, Pedersen RA (2008) Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm. Dev Biol 313:107–117 13. Xu RH, Sampsell-Barron TL, Gu F, Root S, Peck RM, Pan G, Yu J, Antosiewicz-Bourget J, Tian S, Stewart R, Thomson JA (2008) NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. Cell Stem Cell 3:196–206 14. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275–280 15. Singec I, Crain AM, Hou J, Tobe BT, Talantova M, Winquist AA, Doctor KS, Choy J, Huang X, La Monaca E, Horn DM, Wolf DA, Lipton SA, Gutierrez GJ, Brill LM, Snyder EY (2016) Quantitative analysis of human pluripotency and neural specification by in-depth (phospho)proteomic profiling. Stem Cell Reports 7:527–542 16. Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA (2005) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2:185–190 17. Levenstein ME, Ludwig TE, Xu RH, Llanas RA, VanDenHeuvel-Kramer K, Manning D, Thomson JA (2006) Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24:568–574 18. Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS, Llanas RA, Thomson JA (2006) Derivation of human embryonic stem cell in defined conditions. Nat Biotechnol 24:185–187 19. Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA (2006) Feeder-independent culture of human embryonic stem cells. Nat Methods 3:637–646 20. Miyazaki T, Futaki S, Hasegawa K, Kawasaki M, Sanzen N, Hayashi M, Kawase E, Sekiguchi K, Nakatsuji N, Suemori H (2008) Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem Biophys Res Commun 375:27–32

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21. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K, Howden SE, Diol NR, Propson NE, Wagner R, Lee GO, Antosiewicz-Bourget J, Teng JM, Thomson JA (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429 22. Baker DE, Harrison NJ, Maltby E, Smith K, Moore HD, Shaw PJ, Heath PR, Holden H, Andrews PW (2007) Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat Biotechnol 25:207–215 23. Beers J, Gulbranson DR, George N, Siniscalchi LI, Jones J, Thomson JA, Chen G (2013) Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture medium. Nat Protoc 7:2029–2040

24. Lippmann ES, Estevez-Silva MC, Ashton RS (2014) Defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation without small molecule inhibitors. Stem Cells 32:1032–1042 25. Tchieu J, Zimmer B, Fattah F, Amin S, Zeltner N, Chen S, Studer L (2017) A modular platform for differentiation of human PSCs into all major ectodermal lineages. Cell Stem Cell 21:399–410 26. Jacobs K, Zambelli F, Mertzanidou A, Smolders I, Geens M, Nguyen HT, Barbe L, Sermon K, Spits C (2016) Higher-density culture in human embryonic stem cells results in DNA damage and genome instability. Stem Cell Reports 6:330–341

Chapter 6 In Vitro Functional Characterization of Human Neurons and Astrocytes Using Calcium Imaging and Electrophysiology Marita Grønning Hansen, Daniel Tornero, Isaac Canals, Henrik Ahlenius, and Zaal Kokaia Abstract Recent progress in stem cell biology and epigenetic reprogramming has opened up previously unimaginable possibilities to study and develop regenerative approaches for neurological disorders. Human neurons and glial cells can be generated by differentiation of embryonic and neural stem cells and from somatic cells through reprogramming to pluripotency (followed by differentiation) as well as by direct conversion. All of these cells have the potential to be used for studying and treating neurological disorders. However, before considering using human neural cells derived from these sources for modelling or regenerative purposes, they need to be verified in terms of functionality and similarity to endogenous cells in the central nervous system (CNS). In this chapter, we describe how to assess functionality of neurons and astrocytes derived from stem cells and through direct reprogramming, using calcium imaging and electrophysiology. Key words Calcium imaging, Electrophysiology, Neurons, Astrocytes, Stem cells, Reprogramming, Direct conversion

1

Introduction Human neural stem cells (hNSCs) can be derived from fetal brain tissue and subsequently differentiated to the three different neural lineages: neurons, astrocytes, and oligodendrocytes [1]. However, the restricted availability of tissue and ethical implications involved in using human fetal tissue limits their use not only for clinical application but even for research purposes. Since the establishment of human embryonic stem cells (hESCs) [2] and the subsequent discovery that somatic cells, such as fibroblasts, can be reprogrammed to become induced pluripotent stem cells (iPSCs)

Henrik Ahlenius and Zaal Kokaia contributed equally to this work. Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_6, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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[3, 4], numerous protocols have been developed to differentiate hES/iPSCs toward neural lineages [5]. This can be achieved by the continuous differentiation to neurons and/or glia or by isolating intermediary precursors such as NSCs or glial progenitors that can then be terminally differentiated to neurons, astrocytes, and oligodendrocytes. Following the generation of iPSCs, several protocols have also been established to directly convert, bypassing a pluripotent stage, somatic cells toward neural lineages. By overexpressing lineagespecific transcription factors, this approach has been used to generate induced neurons (iNs) [6, 7], neural stem/progenitor cells (iNPC/iNSCs) [8], oligodendroglial cells (iOPCs) [9], and astrocytes (iAstrocytes) [10]. An emerging alternative approach for generating neural cells, and other lineages, employs the logic of direct conversion, using transcription factors, but applying it to hES/iPSCs rather than somatic cells. This transcription factor programming strategy has been used to rapidly and efficiently generate excitatory [11] and inhibitory neurons [12], astrocytes [13] as well as oligodendrocytes [14]. Stem cells, reprogramming, direct conversion, and transcription factor-driven differentiation could all potentially be used to generate neural cells for disease modelling and therapeutic application. However, before considering them for these purposes, they need to be properly evaluated at a functional level. This includes comparisons to their bona fide primary human counterparts. In our laboratories we have extensive experience working with human fetal NSC [15], iPS cell-derived long-term neuroepithelial stem (lt-NES) cells [16, 17], directly converted [18] and transcription factor programming [11, 12]-derived neurons, and astrocytes [13]. We use calcium imaging and electrophysiology routinely to evaluate and compare functional properties of induced cells. Calcium ions play diverse and crucial roles in numerous cellular processes including cell cycle control, cell death, and gene expression in virtually all cells of the body [19]. In the CNS, calcium has even more and critical roles. In neurons, presynaptic influx of calcium stimulates the exocytosis of synaptic vesicles containing neurotransmitters. At postsynaptic dendrites transient elevation of calcium concentration facilitates synaptic plasticity of the neurons [20]. Astrocytes display transient elevations of intracellular calcium both at steady state and in response to various stimuli [21, 22]. Alterations in intracellular calcium in astrocytes have been proposed to signal the release of gliotransmitters that coordinate neuronal activity and to regulate energy supply in the brain [23]. Interrogation of cellular calcium signalling has been made possible by development of fluorescent calcium indicators such as Fluo-4 [24]. Combination of fluorescent calcium indicators with

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real-time confocal imaging is widely used in neuroscience as a first and easy approach to assess functionality of neurons and astrocytes. Electrophysiological characterization of intrinsic and extrinsic properties of neurons differentiated in the culture system is a crucial step in validation of the functionality of these cells. Currently, certain neuronal morphometric characteristics and expression of relevant genes and immunocytochemical markers are necessary, but not sufficient, to consider the given cells as functional neurons of a defined type. This issue becomes extremely important when neurons are derived from hES cells or even more important when neurons are generated from somatic cells through various reprogramming procedures. Patch-clamp recording allows the assessment of passive electrophysiological properties such as membrane potential, input resistance, and capacitance, as well as the ability of presumed neurons to generate action potentials (APs) spontaneously or in response to current-induced depolarizations. The ability to generate an AP is a hallmark of neuronal functionality. Moreover, characteristics of the APs such as threshold, risetime, amplitude, half-height width, and after-hyperpolarization amplitude, together with the presence of sodium and potassium currents, are also important criteria for the validation of the level of functional maturation of neurons. In addition, another functional property of neurons, which can be assessed by patch-clamp electrophysiology, is their ability to receive synaptic input. The presence of synaptic inputs is essential for the neurons to become part of neuronal networks. Astrocytes are by definition non-excitable cells and, thus, do not have the ability to generate an AP. Still, astrocytes have characteristic electrophysiological properties such as hyperpolarized membrane potential and passive membrane currents [25, 26]. Moreover, another functional property of astrocytes, which can be studied by means of patch-clamp electrophysiology, is their support of synapse formation [27]. When setting out to characterize neural cells derived from stem cells or through reprogramming, we typically perform initial validations using calcium imaging, which is then followed by more rigorous electrophysiological assessments. This approach is described below.

2 2.1 2.1.1

Materials Calcium Imaging Equipment

1. Inverted confocal microscope LSM780 (Zeiss) with incubation chamber. 2. ZEN software (Zeiss, 2012 version) with the module for region of interest (ROI) measurement. 3. Glass bottom dishes: Ibidi (81158).

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2.1.2 Solutions

1. Poly-L-ornithine: Sigma (P3655). 2. Phosphate-buffered saline (PBS). 3. Laminin: Sigma (L2020). 4. Poly-D-lysine: Sigma (A-003-E). 5. Fibronectin: Thermo Fisher Scientific (33016015). 6. Fluo-4 AM, FluoroPure™ grade: Molecular probes (F23917). 7. DMSO: Sigma (D2438). 8. Pluronic acid 20% (w/v) in DMSO: Thermo Fisher Scientific (P3000MP). 9. BrainPhys (without phenol red): STEMCELL Technologies (05791). 10. DMEM-F12 (without phenol red): Thermo Fisher Scientific (11039021). 11. Solution pH 4.20 mM sodium acetate, 1 mM CaCl2 dissolved in sterile distilled H2O and adjusted to pH 4. Filter and store at +4
C.

2.2 Electrophysiology 2.2.1 Equipment

1. A fully equipped patch-clamp rig is needed. It should have a recording chamber suitable for coverslips (coverslips with a diameter of 13 mm) and a heating and perfusion system to, respectively, heat up and exchange the recording solution continuously. 2. Glass capillaries with filament (King Precision Glass or similar), 85 mm long, with 0.86 mm inner and 1.50 mm outer diameters, are needed for the patch pipettes. 3. Pipette puller (P-97 Sutter pipette puller, Sutter Instrument, or similar), with the ability to pull patch pipettes. 4. Sectioning vials (CERB111040, VWR) for fixation of cells after recordings.

2.2.2 Solutions

1. Artificial cerebrospinal fluid (ACSF): 119 mM NaCl, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 1.3 mM MgSO4, and 11 mM glucose are dissolved in Milli-Q and bubbled with carbogen for 15 min before adding 2.5 mM CaCl2. pH ~7.4 and osmolarity ~307 mOsm. Keep at room temperature (RT). 2. Potassium gluconate-based intracellular solution (KGlu intra): 122.5 mM KGlu, 12.5 mM KCl, 10 mM HEPES, 2 mM Na2ATP, 0.3 mM Na2GTP, 2 mM MgCl2, and 8.0 mM NaCl. Adjust pH to 7.3 with KOH. Osmolarity ~300 mOsm. Dissolve 1–3 mg/mL biocytin (epsilon-biotinoyl-L-lysine, Mw ¼ 372.48 g/mol, Biotium) in the pipette solution prior recording.

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3. Cesium chloride-based intracellular solution (CsCl intra): 135 mM CsCl, 10 mM NaCl, 10 mM HEPES, 2 mM Na2ATP, 0.3 mM Na2GTP, and 2 mM MgCl2. Adjust pH to 7.3 with CsOH. Osmolarity ~290 mOsm. Dissolve 1–3 mg/ mL biocytin (epsilon-biotinoyl-L-lysine, Mw ¼ 372.48 g/mol, Biotium) and 1.72 mg/mL QX314 bromide (N-Ethyllidocaine bromide, Mw ¼ 343.31 g/mol, Abcam Biochemicals) in the pipette solution prior recording. 4. TTX (tetrodotoxin, citrate free, Mw ¼ 319.28 g/mol, Latoxan), 0.2 mM stock solution. Dissolve 1 mg TTX in 15.66 mL Milli-Q added 10 μL 1 M HCl to facilitate dissolution. Aliquot into 0.5 mL vials and store in the freezer. Use 1 μM TTX in ACSF for recordings. 5. TEA (tetraethylammonium chloride, Mw ¼ 183.72 g/mol, Abcam Biochemicals), 1 M stock solution. Dissolve 4.593 g TEA in 25 mL Milli-Q. Aliquot into 1 mL vials and store in the freezer. Use 10 mM TEA in ACSF for recordings. 6. PTX (picrotoxin, Mw ¼ 602.59 g/mol, Abcam Biochemicals), 100 mM stock solution. Dissolve 301.295 mg PTX in 5 mL DMSO. Aliquot into 200 μL vials and store in the freezer (see Note 1). Use 100 μM PTX in ACSF for recordings. 7. D-AP5 (Mw ¼ 197.13 g/mol, Abcam Biochemicals), 0.1 M stock solution. Dissolve 50 mg D-AP5 in 2.536 mL 0.1 M NaOH. Aliquot into 100 μL vials and store in the freezer. Use 50 μM D-AP5 in ACSF for recordings. 8. NBQX (Abcam Biochemicals) (see Note 2), 5 mM stock solution. Dissolve 10 mg NBQX in 2/Mw (NBQX) L (see Note 3) Milli-Q. Aliquot into 200 μL vials and store in the freezer. Use 5 μM NBQX in ACSF for recordings. 9. PFA (paraformaldehyde, 95%, Mw ¼ 30.03 g/mol, SigmaAldrich), 4% solution. Total volume of 1 L. Preheat 500 mL 0.2 M NaOH to just below 60
C. Add 40 g of PFA (in the chemical hood), and mix until the PFA has dissolved. Add 380 mL 0.2 M Na2HPO4 (Na2HPO4  2H2O,  99.0%, Mw ¼ 177.99 g/mol, Sigma) and 120 mL 0.2 M NaH2PO4 (NaH2PO4  H2O,  99.5%, Mw ¼ 137.99 g/mol, Sigma). Filter the solution and leave to cool down. Adjust pH to 7.4. Aliquot into 10 mL vials and freeze. Store at 4
C after thawing. Fix cells in 1 mL of 4% PFA. 10. Triton™X-100, Sigma (T8787). 11. PVA mounting media with DABCO, (1,4-diazabicyclo-(2,2,2) octane, Sigma, D2522). Dissolve 4.8 g of PVA (polyvinyl alcohol, Sigma, P8136) in 12 g of glycerol and mix. Add 12 mL of Milli-Q water, and mix overnight at RT using a rotator. Add 24 mL of 0.2 M Tris–HCl at pH 8–8.5, and mix in a water bath at 50
C for 30 min. Add 1.25 g of DABCO and

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mix well. Centrifuge at 5000  g for 15 min, and remove the supernatant. Aliquot and store at 20
C. 12. Normal donkey serum (Sigma, S30-M).

3 3.1

Methods Cell Preparation

1. Culture cell type of interest according to previously established methods then proceed as described below. 2. Coating. (a) For neurons: Poly-L-ornithine (0.1 mg/mL in Milli-Q) is added to the surface of glass bottom dishes or coverslips and incubated overnight at RT. Remove the poly-L-ornithine solution and rinse twice with PBS. Add enough laminin (10 μg/mL in PBS) to cover the surface and incubate at +37
C overnight. (b) For astrocytes: Poly-D-lysine (0.1 mg/mL in Milli-Q) is added to the surface of glass bottom dishes or coverslips and incubated 2 h at RT. Remove the poly-D-lysine solution and rinse twice with PBS. Add enough fibronectin (10 μg/mL in PBS) to cover the surface, and incubate at +37
C overnight. (c) For co-cultures: Poly-D-lysine (0.1 mg/mL in Milli-Q) is added to the surface of glass bottom dishes or coverslips and incubated 2 h at RT. Remove the poly-D-lysine solution and rinse twice with PBS. Add enough laminin (10 μg/mL in solution pH 4) to cover the surface, and incubate at 37
C overnight. 3. Plate neurons or astrocytes separately or in combination on coated coverslips or glass bottom dishes. Density of cells will vary depending on the application but is in general 100,000–250,000 cells per glass bottom dish and 50,000–100,000 neurons and/or 40,000–50,000 astrocytes per coverslip. When plating astrocytes for mechanical stimulation, consider high-density cultures since spreading of intracellular calcium raises is depending on cell-to-cell contacts.

3.2

Calcium Imaging

3.2.1 Calcium Loading and Imaging of Neurons and Astrocytes

1. One vial (50 μg) of the calcium dye Fluo-4 (for other dyes see Table 1) is reconstituted in 25 μL of DMSO and 25 μL of pluronic acid (stock solution 1 mg/mL). This stock can be stored at 20
C for 1–2 months. 2. Working solution is prepared freshly in the hood and protected from light by diluting the calcium dye 1:1000 (final concentration of dye 1 μg/mL) in cell media without phenol red. 3. Approximately 2 mL of calcium dye working solution is applied on differentiated cells plated on glass bottom dishes compatible

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Table 1 Different calcium indicators Calcium indicator

Excitation (nm)

Emission (nm)

Kd (nM)

Comments

Calcium Green

488

525

190

Fluo-3

506

526

325

Fluo-4

494

516

345

Fura-2

340/380

510

140

Ratiometric

Indo-1

338

400/475

230

Ratiometric, high photobleaching

Oregon Green

488

520

170

Rhod-2

556

576

570

X-rhod-1

580

602

700

with fluorescence-inverted microscope for live-cell imaging (see Note 4). 4. Cells are incubated at 37
C in the dark, i.e., an incubator, for at least 25 min. 5. Calcium dye working solution is removed from the cells, and fresh media (2 mL) without phenol red is added to the dish. 6. The dish with the loaded cells is immediately transferred to the microscope (see Note 5). 7. Fluorescent images from one field of view with good ratio between number of cells and resolution (see Note 6) are taken every second during the time of the experiment (10–30 min) (see Note 7). Depending on the exposure time, latency between pictures can be from 1 to 5 s. More than 5 s is not recommended since fast calcium transitions can be missed. 8. Basal activity is recorded for at least 2 min (see Figs. 1a and 2a). Depending on the experiment and the kind of cells, different stimuli can be applied, such as neurotransmitters or mechanical stimuli (see Table 2, Figs. 1b and 2b). 3.2.2 Analysis of Neurons

1. The somas of the cells in the frame are defined using the ROI measurement tool from ZEN software (Zeiss, 2012 version). 2. Profile of fluorescence intensity for each cell over time is generated. 3. Intensity profile should be normalized as follows: every single measurement for each cell at the different time points is divided by a constant, which can be the average during a period of time without activity (firing) or the lowest level of fluorescence for that particular cell. An increase in the ratio exceeding fluctuation of the basal level should be considered an event (this has to

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C

D Ratio (AU)

B B

Ratio (AU)

A A

Fig. 1 (a, b) Human iPS-derived neurons in culture loaded with Fluo-4 before (a) and after glutamate stimulation (b). Most of the neurons responded with an increase in intracellular calcium observed as higher fluorescence levels. (c) Analysis of three neurons with spontaneous activity. (d) Analysis of three neurons that responded to addition of glutamate represented by the arrow. Note the difference in the scale of the fluorescence axis between spontaneous activity (c) and stimulated response (d). Ratio in arbitrary units (AU) shows the fluorescence at each time point in relation to the lowest fluorescence value for each cell

be defined by the researcher based on the profile of each cell). A singular increase in magnitude or frequency of events after stimulus should be considered as a response. 4. Different information that can be obtained: (a) Spontaneous activity (see Fig. 1c). (b) Frequency of events. (c) Yes or no response to stimulus (see Fig. 1d). 3.2.3 Analysis of Astrocytes

1. Define the somas of the cells in the frame using the ROI tool from ZEN software (Zeiss, 2012 version). 2. Profile of fluorescence intensity over time for each cell is automatically generated by ZEN software.

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C

D

Ratio (AU)

B B

Ratio (AU)

A A

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Fig. 2 (a, b) Astrocytes in culture loaded with Fluo-4 before (a) and after (b) ATP stimulation. A clear increase in fluorescence levels can be detected in most of the cells. (c) Astrocytes that did not show any spontaneous activity during 5 min showed different responses to ATP stimulation. (d) Astrocytes that showed spontaneous intracellular calcium raises were also responding to ATP stimulation. Ratio (AU) shows the fluorescence at each time point in relation to the basal fluorescence value (average of the first 2 min without stimulation)

3. Basal fluorescence intensity is used to normalize recording data at each time point. Basal level is considered as the average intensity when cells are not having spontaneous calcium rises during the first minutes of recording, before any stimulus is applied. Alternatively, basal level could be considered as the lowest value before addition of stimulus. 4. An increase in the fluorescence exceeding 50% of the basal level should be considered as an event (spontaneous activity or positive response to stimulus). 5. Different information that can be obtained: (a) Spontaneous activity (see Fig. 2d) to determine if cells present intracellular calcium rises without stimulus. (b) Frequency of response (events; see Fig. 2c, d) as the number of calcium rises over time before or after stimulus.

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Table 2 Examples of means of stimulating neurons and astrocytes Stimulus

Type

Concentration

Cells

Glutamate

Neurotransmitter

0.03–100 μM

Neurons and astrocytes

ATP

Nucleotide

0.03–100 μM

Astrocytes

KCl

Ionic

30–100 mM

Neurons and astrocytes

GABA

Neurotransmitter

100 μM

Neurons

ACh

Neurotransmitter

100 μM

Neurons and astrocytes

Nicotine

Neurotransmitter

100 μM

Neurons

NMDA

Agonist

3–100 μM

Neurons

AMPA

Agonist

3–100 μM

Neurons

Manual stimulation

Mechanical

Astrocytes

(c) Percentage of cells responding to molecular or mechanical stimulus by an increase in intracellular calcium (see Fig. 2a–d). 3.3 Electrophysiology

1. A coverslip with the cells of interest attached is transferred to the patch-clamp setup and continuously perfused with ACSF (1–2 mL/min) at +34
C. 2. Whole-cell patch-clamp recordings are performed. Patch pipettes with resistance of 3–7 MΩ are used for patching. Patch pipettes are filled with KGlu-intracellular solution unless otherwise specified. The intracellular solution contains 1–3 mg/ mL biocytin for post hoc identification of the cell. Approach the cell and obtain a gigaseal before opening the cell. 3. After gaining access to the inside of the cell, the series resistance (Rseries) and input resistance (Rinput) are determined in voltage clamp by means of a 50-ms-long 5 or 10 mV test pulse from a holding potential of 70 mV (see Note 8). The protocol is repeated 10 times with 1 s interval. 4. Determine the resting membrane potential in current clamp mode without injecting any current for 0.5–1 min.

3.3.1 Neuronal Characteristics

1. To determine the neurons’ ability to fire action potentials (APs) and to generate a current-voltage plot, keep the cell in current clamp mode, and adjust the membrane potential to be around 70 mV by injecting current. Induce APs by current injections, 10 pA steps from 0 to 390 pA lasting 500 ms with an interval of 2 s.

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2. To determine the AP characteristics, keep the cell in current clamp mode, and adjust the membrane potential to be around 70 mV by injecting current (see Note 9). Induce APs by means of a current ramp, lasting 1 s, going from 0 to 300 pA or 0 to 500 pA. Use the first AP generated during the current ramp to determine the AP characteristics. 3. The presence of sodium and potassium currents is determined in voltage clamp mode. The cell is kept at 70 mV. Voltage steps of 10 mV, ranging from 70 mV to +40 mV and lasting 200 ms, are executed with 2 s intervals. 4. Perfuse the cells with ACSF containing 1 μM TTX. Repeat steps 2 and 3 to show that the APs and the sodium current are blocked by TTX. 5. Perfuse the cells with ACSF containing 1 μM TTX + 10 mM TEA. Repeat step 3 to show that the outward sustained potassium currents are reduced (see Note 10). 3.3.2 Synaptic Connections Between Neurons

1. In order to assess synaptic connections, it is crucial that neurons and astrocytes have been co-cultured as presence of astrocytes greatly increases synapse formation. 2. Whole-cell patch-clamp recordings are performed with a CsClbased intracellular solution. Hold the cell at 70 mV in voltage clamp mode, and record continuously for 2–3 min to identify spontaneous postsynaptic currents (sPSCs) (see Note 11). If sPSCs are observed, continue to step 2 or 3 to study glutamatergic or GABAergic sPSCs, respectively. 3. In the case of glutamatergic sPSCs, exchange the ACSF with ACSF + 100 μM PTX to isolate the glutamatergic sPSCs. Record sPSCs for 2–3 min at a holding potential of 70 mV. Block the glutamatergic sPSCs by application of 50 μM D-AP5 and 5 μM NBQX to the ACSF + 100 μM PTx. Record sPSCs for another 2–3 min. 4. In the case of GABAergic sPSCs, exchange the ACSF with ACSF + 50 μM D-AP5 + 5 μM NBQX to isolate the GABAergic sPSCs. Record sPSCs for 2–3 min at a holding potential of 70 mV. Block the GABAergic sPSCs by application of 100 μM PTX to the ACSF + 50 μM D-AP5 + 5 μM NBQX. Record sPSC for another 2–3 min.

3.3.3 Functional Characteristics of Astrocytes

1. To determine the linearity of the current-voltage relationship of astrocytes, keep the cell in voltage clamp mode, and hold the cell at 80 mV. Use a voltage step protocol lasting 50 ms and going from 170 mV to positive voltages (range between +10 mV and + 60 mV) in 10 mV steps with 2 s between voltage steps to induce voltage-dependent currents.

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3.3.4 Astrocytic Support of Synapse Formation

1. Co-culture neurons with and without astrocytes (see Note 12) to be able to determine the ability of the astrocytes to support synapse formation between neurons. 2. Perform whole-cell patch-clamp recordings from neurons to detect the presence of sPSC in a similar manner as described in Subheading 3.3.2.

3.3.5 Post Hoc Identification of Cells

1. By the end of the recording, which should last at least 5 min to assure sufficient diffusion of biocytin into the cell, slowly remove the patch pipette while applying a small amount of positive pressure to remove the cell membrane from the pipette tip and to leave the cell intact on the coverslip. 2. When finished with the recordings, place the coverslip in a sectioning vial with 1 mL 4% PFA at +5
C. Leave for fixation at least 20 min. 3. Rinse coverslips three times with PBS. 4. Permeabilize and block using 0.025% Triton™ X-100 with 5% normal donkey serum in PBS for 1 h at RT. 5. Incubate cells at +4
C overnight with primary antibodies (for detection of neurons/astrocytes) diluted in blocking solution. 6. Apply fluorophore-conjugated secondary antibodies diluted in blocking solution for 2 h at RT. 7. Rinse with PBS, and incubate the cells with Cy3-conjugated streptavidin diluted in PBS for 30 min at RT. 8. Rinse twice with PBS, and counterstain with Hoechst diluted in PBS for 10 min at RT. 9. Rinse with PBS and mount with DABCO. 10. Take pictures using epifluorescence microscope to identify biocytin-positive cells. 11. Patched cells are filled with biocytin, and the morphology of the patched cells is visible after the staining. In recordings from neurons, only patched neurons are biocytin positive. In recordings from astrocytes, where biocytin can diffuse via gap junctions, both the patched astrocyte and neighboring astrocytes, which are connected to the patched astrocyte via gap junctions, are positive for biocytin.

3.3.6 Analysis of the Basic Characteristics

1. Determination of Rseries and Rinput: Align the baseline for 10 current traces induced by a 50 ms long 5 or 10 mV test pulse. Make an average of the ten traces, and use the averaged trace to determine Rseries and Rinput. The amplitude of the downward deflecting current peak, Iseries, is used to calculate Rseries, and the steady state current, Iinput, determined as the average current at 40–45 ms into the 50 ms test pulse, is used

Functional Characterization of Neurons and Astrocytes

A

B

C K current

Amplitude

Peak Iseries

1/2 AP amplitude width Threshold AHP

20ms

D

5ms

Glutamatergic sPSC

0.5nA

-40mV

20mV

200pA

Iinput

85

20ms

GABAergic sPSC

50ms

Na current

E

2ms

0.5nA

20pA

100pA 5ms

1.5

0.5nA

Current (nA)

1.0 0.5 0.0 -0.5 -1.0 -150

-170mV to +10mV

-100 -50 Voltage (mV)

0

10ms

Fig. 3 (a) Averaged current trace induced by a 50 ms long 10 mV test pulse. Dashed lines indicate Iseries and Iinput. (b) Voltage trace of a ramp-induced AP. Measurements of AP threshold, amplitude, peak, AHP, and one-half AP amplitude width are illustrated. (c) Current trace induced by a 200 ms long voltage step. The fast inward sodium current is indicated by an arrow. The dashed lines indicate the outward sustained potassium current. (d) Example current traces of a glutamatergic sPSC and a GABAergic sPSC. (e) Current traces induced by 50 ms long 10 mV big voltage steps ranging from 170 to +10 mV. Dashed lines indicate the measured current (left). Plot illustrates the current-voltage relationship (right)

to calculate Rinput (Fig. 3a). The resistances are calculated by means of Ohm’s law: R¼

V I

where V is the voltage step (5 or 10 mV) and I determined from the averaged current trace. 2. Determination of the capacitance, C: The decay time, τ, of the test pulse-induced current is determined and used to calculate the capacitance: C¼

τ R

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3.3.7 Analysis of the Functional Characteristics of Neurons

1. Ability to generate APs: The number of APs generated at all step current injections from 0 to 390 pA is noted. The number of APs is plotted as a function of the current injection. The maximal number of APs generated for each cell is noted and used for comparison. 2. AP characteristics: The AP threshold is detected at the onset of the AP. The AP amplitude is determined as the voltage from AP threshold to AP peak. The risetime can be measured as the time from AP threshold to AP peak. The afterhyperpolarizing potential (AHP) can be detected as the voltage difference between the AHP peak and the AP threshold. The half AP amplitude width is also determined (Fig. 3b). 3. Presence of Na current: Identify and measure the fast inward current present in the current traces induced by 10 mV voltage steps from 70 to +40 mV (Fig. 3c). Plot the current peak as a function of the voltage. Repeat the measurement for the recordings performed in the presence of TTX, and plot together with the other graph. 4. Presence of outward sustained K current: Identify and measure the sustained outward current present in the current traces induced by 10 mV voltage steps from 70 to +40 mV (Fig. 3c). Plot the current peak as a function of the voltage. Repeat the measurement for the recordings performed in the presence of TTX + TEA, and plot together with the other graph.

3.3.8

Analysis of sPSCs

1. Detection limits: Align the baseline of the 2–3-min-long continuous current trace, used for detection of sPSCs, to zero. Determine the noise levels of the control trace, and prepare a noise histogram, and determine the standard deviation (SD) of the noise. The threshold for detection of sPSCs is set to 3  SDnoise. 2. Detection criteria: sPSCs are identified as events with an amplitude of at least 3  SDnoise and a risetime that is faster than the decay time of the events. In the experimental settings used here, the glutamatergic events are fast decaying, while the GABAergic events decay slowly (Fig. 3d). Determine the frequency of events and the averaged amplitude and decay time. Use this for comparison.

3.3.9 Analysis of the Functional Characteristics of Astrocytes

1. Current-voltage relationship: Plot the current as a function of the voltage steps, and determine the linearity of the graph (Fig. 3e).

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1. Determine the presence of sPSCs, the frequency of sPSCs, and the averaged amplitude and decay time, as described in Subheading 3.3.8. 2. Compare the occurrence, frequency, amplitude, and decay time of sPSCs detected in neurons, either cultured alone or in the presence of astrocytes, to assess whether astrocytes have the ability to support synapse formation.

4

Notes 1. For experimental concentration of PTX at 0.1 M, DMSO in the final solution is equal or less than 0.1% . 2. The molecular weight and color of NBQX vary from batch to batch. 3. V ¼ m/(C  Mw) ¼ 10 mg/(5 mM  Mw(NBQX)) ¼ 2gL/ mol/Mw(NBQX), i.e., if Mw(NBQX) ¼ 398.26 g/mol, V ¼ 2gL/mol/398.26 g/mol ¼ 0.005022 L ¼ 5.022 mL milliQ. 4. Alternatively cells can be plated on coverslips that then is mounted into a chamber suitable for inverted microscope. 5. Controlled temperature at +37
C, 5% CO2, and humidity are desirable, especially for long-term recordings. 6. It is desirable to have at least 20 cells in the field; so depending on the density, 10 or 20 objective can be used. 7. In case of confocal microscope, pinhole should be high to increase the amount of light and reduce the exposure time. No z-stacks are needed since we are dealing with cells in monolayer. 8. The series resistance should be below 30 MΩ. 9. Ideally, maximal 50 pA of current injection is needed to keep the cell at 70 mV. 10. The series resistance should remain stable throughout the experiment, as the current amplitudes are altered if the series resistance changes. Thus, it is important to determine the series resistance multiple times during the experiments, especially, before and after drug application. 11. Under the specified recording conditions, postsynaptic currents are identified as fast onset, downward deflecting currents, which decay exponentially. Glutamatergic events decay fast, while GABAergic events have a slow decay time. 12. Ideally, use co-cultures of neurons with, i.e., mouse astrocytes as a positive control.

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References 1. Uchida N et al (2000) Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 97(26):14720–14725 2. Thomson JA et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145–1147 3. Takahashi K et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872 4. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676 5. Tao Y, Zhang SC (2016) Neural subtype specification from human pluripotent stem cells. Cell Stem Cell 19(5):573–586 6. Pang ZP et al (2011) Induction of human neuronal cells by defined transcription factors. Nature 476(7359):220–223 7. Vierbuchen T et al (2010) Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463(7284):1035–1041 8. Lujan E et al (2012) Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells. Proc Natl Acad Sci U S A 109(7):2527–2532 9. Yang N et al (2013) Generation of oligodendroglial cells by direct lineage conversion. Nat Biotechnol 31(5):434–439 10. Caiazzo M et al (2015) Direct conversion of fibroblasts into functional astrocytes by defined transcription factors. Stem Cell Rep 4 (1):25–36 11. Zhang Y et al (2013) Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron 78(5):785–798 12. Yang N et al (2017) Generation of pure GABAergic neurons by transcription factor programming. Nat Methods 14(6):621–628 13. Rapid and efficient induction of functional astrocytes from human pluripotent stem cells. Canals I, Ginisty A, Quist E, Timmerman R, Fritze J, Miskinyte G, Monni E, Hansen MG, Hidalgo I, Bryder D, Bengzon J, Ahlenius H. Nat Methods. 2018 Sep;15(9):693–696. https://doi.org/10.1038/s41592-018-01032. Epub 2018 Aug 20. PMID: 30127505 14. Ehrlich M et al (2017) Rapid and efficient generation of oligodendrocytes from human

induced pluripotent stem cells using transcription factors. Proc Natl Acad Sci U S A 114(11): E2243–E2252 15. Kallur T et al (2006) Human fetal cortical and striatal neural stem cells generate regionspecific neurons in vitro and differentiate extensively to neurons after intrastriatal transplantation in neonatal rats. J Neurosci Res 84 (8):1630–1644 16. Tornero D et al (2017) Synaptic inputs from stroke-injured brain to grafted human stem cell-derived neurons activated by sensory stimuli. Brain 140(3):692–706 17. Tornero D et al (2013) Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery. Brain 136 (Pt 12):3561–3577 18. Miskinyte G et al (2017) Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks. Stem Cell Res Ther 8(1):207 19. Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1(1):11–21 20. Grienberger C, Konnerth A (2012) Imaging calcium in neurons. Neuron 73(5):862–885 21. Newman EA, Zahs KR (1997) Calcium waves in retinal glial cells. Science 275 (5301):844–847 22. Parpura V et al (1994) Glutamate-mediated astrocyte-neuron signalling. Nature 369 (6483):744–747 23. Bazargani N, Attwell D (2016) Astrocyte calcium signaling: the third wave. Nat Neurosci 19(2):182–189 24. Paredes RM et al (2008) Chemical calcium indicators. Methods 46(3):143–151 25. Bedner P et al (2015) Astrocyte uncoupling as a cause of human temporal lobe epilepsy. Brain 138(Pt 5):1208–1222 26. Dallerac G, Chever O, Rouach N (2013) How do astrocytes shape synaptic transmission? Insights from electrophysiology. Front Cell Neurosci 7:159 27. Chung WS, Allen NJ, Eroglu C (2015) Astrocytes control synapse formation, function, and elimination. Cold Spring Harb Perspect Biol 7 (9):a020370

Chapter 7 Differentiation of Neural Stem Cells Derived from Induced Pluripotent Stem Cells into Dopaminergic Neurons Marcel M. Daadi Abstract Dopaminergic (DA) neurons are involved in many critical functions within the central nervous system (CNS), and dopamine neurotransmission impairment underlies a wide range of disorders from motor control deficiencies, such as Parkinson’s disease (PD), to psychiatric disorders, such as alcoholism, drug addictions, bipolar disorders, schizophrenia and depression. Neural stem cell-based technology has potential to play an important role in developing efficacious biological and small molecule therapeutic products for disorders with dopamine dysregulation. Various methods of differentiating DA neurons from pluripotent stem cells have been reported. In this chapter, we describe a simple technique using dopamineinducing factors (DIFs) to differentiate neural stem cells (NSCs), isolated from induced pluripotent stem cells (iPSCs) into DA neurons. Key words Self-renewable neural stem cells, iPSCs, Dopaminergic neuron differentiation

1

Introduction Parkinson’s disease (PD) is the second most common chronic neurodegenerative disease after Alzheimer’s. Approximately one million people in the USA suffer from PD with an economic burden of $6 billion annually, and its prevalence is expected to grow as the general population ages. Cell therapy for PD has shown promising outcome in preclinical and clinical studies [1–28]. However, there is a lack of proven technology to develop and commercialize a viable dopaminergic (DA) neuron product derived from pluripotent human embryonic stem cells (hESCs) or from induced pluripotent stem cells (iPSCs). The safety and efficacy of neural stem cell (NSC)-derived DA neurons manufactured and cryopreserved under conditions amenable to clinical use and commercialization are unknown. The production of a clinical grade neural cell product requires meeting and overcoming numerous challenges in process development and optimizing critical steps. These steps include establishing a scalable platform from

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_7, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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the beginning to minimize comparability risks and addressing upfront issues regarding harvest, formulation, filling, cryopreservation, and storage to enable product development. We have reported a process that generates DA neurons from NSCs derived from various sources, including mouse brain, human neural tissue (from embryonic and adult brain cells), and pluripotent hESCs. Here we describe a method for generating DA neurons from human iPSCs. The technology uses dopamine-inducing factors (DIFs) and basic fibroblastic growth factor (FGF2), which together induce the midbrain DA phenotype in NSCs. Other technologies use the combination of sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) to derive dopaminergic neurons from mouse embryonic stem cells (ESCs) [29, 30]. The derivation of DA neurons from hESCs has been achieved by exposure to SHH, FGF8, and small molecules, such as CHIR [9] activators of the Wnt signaling pathway, and by co-culturing with feeder cell layers, such as PA6 or amniotic membrane matrix, or through the derivation of floor plate precursors [6, 9, 15, 22, 31–41]. However, so far these technologies present challenging issues in manufacturing and downstream processing of the final product and present obstacles to moving promising cell lines with therapeutic and commercial potential into the clinical arena. The simple method we describe here provides an alternative approach to generate an unlimited supply of DA neurons from self-renewable NSCs for stem cell basic biology, drug screening, and cell therapy (Fig. 1).

2 2.1

Materials Equipment

1. Cell culture incubator (Nuaire, Plymouth, MN, USA). 2. Phase-contrast microscope (Zeiss, Oberkochen, Germany). 3. Centrifuge (Eppendorf, Hamburg, Germany). 4. T25 tissue culture flask (Corning, Oneonta NY, USA). 5. T75 tissue culture flask (Corning, Oneonta NY, USA). 6. Cell lifter (Fisher scientific, Pittsburgh, PA, USA). 7. Glass pipettes (Fisher scientific, Pittsburgh, PA, USA). 8. Centrifuge tubes (15 and 50 mL) (Corning, Oneonta NY, USA). 9. Syringe filters (Corning, Oneonta NY, USA). 10. Syringes (10, 20, and 60 mL) (BD, Franklin Lakes, NJ, USA). 11. Pipettes (2–25 mL) (Fisher scientific, Pittsburgh, PA, USA). 12. Pipette aids (10–1000 μL) (Eppendorf, Hamburg, Germany). 13. Pipette tips (10–1000 μL) (Accuflow, E&K Scientific, Santa Clara, CA, USA).

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Fig. 1 Generation of dopaminergic neurons from iPSC-derived NSCs. (a) Phase-contrast image showing example of differentiated NSCs. (b) Photograph showing that NSCs respond to dopamine-inducing media DIF1 and express the tyrosine hydroxylase marker for DA neurons (TH, red) and the neuronal marker TuJ1 (green). (c) Graph showing results of quantitative polymerase chain reaction (Q-PCR) analysis performed on DIF1 differentiated NSCs for selected transcription factors confirming the dopaminergic identity of the differentiated NSC progeny

14. Water bath (Fisher scientific, Pittsburgh, PA, USA). 15. Hemocytometer (Hausser Scientific, Horsham, PA, USA) or automated cell counter (Countess, Invitrogen, Carlsbad, CA, USA). 2.2

Reagents

1. NN1 media (Neoneuron, San Antonio, TX, USA). 2. DIF1 (Neoneuron, San Antonio, TX, USA). 3. Basic fibroblast growth factor (Stemgent, Cambridge, MA, USA). 4. Poly-L-ornithine hydrobromide (Sigma-Aldrich, St. Louis, MO, USA).

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5. Fetal bovine serum (FBS, GE, Logan, Utah, USA). 6. Ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA). 7. Accutase (Gibco, Life Technologies, NY, USA). 8. Trypsin neutralizer (Gibco, Life Technologies, NY, USA). 9. Tris base (Fisher Bioreagents, Pittsburgh, PA, USA). 10. Phosphate buffered saline (PBS, Gibco, Life Technologies, NY, USA). 11. Double-distilled water (Gibco, Life Technologies, NY, USA).

3

Methods

3.1 Preparation of Reagents and Media: Prepare all the Reagents Under Sterile Conditions in a Horizontal Laminar Flow Hood

1. Preparation of 10 mM Tris (25 mL): Dissolve 30.35 mg of Tris base (F.Wt: 121.4) in 15 mL of double-distilled water, and adjust the pH to 7.6. Make up the volume to 25 mL and filter sterilize. Store at 4  C (see Note 1). 2. Preparation of basic fibroblast growth factor stock solution: Briefly centrifuge the tube, and reconstitute the bFGF in 2.5 mL of 10 mM Tris solution (pH 7.6) to prepare a 20 μg/mL stock solution. Aliquot and store at 20  C. 3. Preparation of L-ascorbic acid stock solution (200 mM): Dissolve 352.24 mg of L-ascorbic acid (Sigma, F.wt: 176.12) in 10 mL of double-distilled water to achieve a final concentration of 200 mM. Filter, sterilize, aliquot, and store at 20  C. 4. Preparation of differentiation media-A (50 mL): Add 37.5 mL of DIF1 (75% V/V), 12.5 mL of NN1 media (25% V/V), 25 μL of bFGF stock solution (final concentration 10 ng/ mL), 50 μL of L-ascorbic acid stock solution (final concentration 200 μM), and 500 μL of FBS (final concentration 1%). Filter, sterilize, and store at 4  C (see Note 2). 5. Preparation of differentiation media-B (50 mL): Add 12.5 mL of DIF1 (25% V/V), 37.5 mL of NN1 media (75% V/V), 25 μL of bFGF stock solution (final concentration 10 ng/ mL), 50 μL of L-ascorbic acid stock solution (final concentration 200 μM), and 500 μL of FBS (final concentration 1%). Filter sterilize, and store at 4  C (see Note 2). 6. Poly-L-ornithine hydrobromide (PLO): Dissolve poly-l-ornithine hydrobromide (PLO) in DD water under sterile conditions to prepare a 10 stock solution of 0.16 mg/mL. The stock solution can be aliquoted and stored at 20  C for future use.

3.2 Coating Culture Plates with PLO

1. Under sterile conditions, prepare working solution of PLO (16 μg/mL) by diluting 1 mL of 10 stock solution (0.16 mg/mL) in 9 mL of DD water.

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2. Add PLO working solution to each well of the desired plate type (6–96 well) to cover the entire surface. We typically use the following volumes for each well for the following type of cell culture plates: Plate type

Volume (μL)

96

25–30

48

80

24

250

12

500

6

1000

3. Incubate the plate in the cell culture incubator for a minimum of 2 h. 4. After incubation wash the plate twice with sterile PBS and plate the cells. The plates can be prepared a day in advance and stored 4  C under sterile culture conditions. 3.3 Differentiation of iPSC-Derived NSCs: For Generation and Expansion of NSCs from iPSCs, Please Refer to Chap. 1

1. Prewarm the differentiation media-A in a 37  C water bath. 2. Collect the floating neurospheres (day 7 of culture, refer Chap. 1) from the T-75 flask into a 50 mL tube, and centrifuge at 1000 rpm (200  g) for 5 min. 3. Carefully remove the supernatant by using vacuum suction without disturbing the pellet. 4. Gently resuspend the pellet in a 10 mL of fresh NN1 media, and centrifuge again at 1000 rpm (200  g) for 5 min. 5. Resuspend the neurospheres in differentiation media-A. 6. Plate the neurospheres on PLO-coated plates. We typically use the following volumes for each well: Plate type

Volume (μL)

96

100

48

250

24

500

12

1000

6

2000

7. Day 0: The day of plating the neurospheres with the differentiation media-A. 8. Change media every 24 h for the next 2 days (day 1 and day 2) with differentiation media-A.

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9. On day 3 or 72 h after plating, switch the cells to differentiation media-B. 10. The cells can be maintained in differentiation media-B until the completion of the experiment or the day of transplantation. 3.4 Harvesting the Differentiated DA Neurons Derived from NSCs for Transplantation: Depending on the Study Requirements, the DA Neurons Can Be Harvested Starting from Day 7 of Differentiation

1. Prewarm PBS, accutase, and trypsin neutralizer in a 37  C water bath. 2. Carefully remove the media from the wells, and wash the cells once with sterile PBS. 3. Add 500 μL of accutase to each well of a 6 well plate, and incubate for 3 min at 37  C in a cell culture incubator. 4. After 3 min stop the activity of accutase by adding trypsin neutralizer. 5. Gently triturate the cells to prepare a single-cell suspension, and transfer the suspension to a 15 mL tube. 6. Centrifuge at 1000 rpm (200  g) for 5 min. 7. Carefully remove the supernatant by using vacuum suction without disturbing the pellet. 8. Resuspend the pellet in 5 mL of sterile PBS. 9. Count the cells using either hemocytometer or automated cell counter using trypan blue exclusion. Calculate the percentage viability and total number of the cells. Record in the lab notebook. 10. Centrifuge the cell suspension at 1000 rpm (200  g) for 5 min, and resuspend in PBS to prepare a 100,000 cells/μL cell suspension. 11. Hold the cells on ice until transplantation.

4

Notes 1. Prepare all the solutions and reagents in double-distilled water or ultrapure water (18 MΩ cm at 25  C). 2. Calculate the amount of differentiation media required for the experiment in advance, and prepare the media. This will help to maintain the constant concentration of all the factors during differentiation.

Acknowledgment This work was supported by NeoNeuron LLC. Disclosures: Dr. Marcel M. Daadi is founder of the biotech company NeoNeuron.

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References 1. Isacson O (2003) The production and use of cells as therapeutic agents in neurodegenerative diseases. Lancet Neurol 2(7):417–424 2. Brazel CY, Rao MS (2004) Aging and neuronal replacement. Ageing Res Rev 3(4):465–483 3. Bjo¨rklund A et al (1987) Mechanisms of action of intracerebral neural implants: studies on nigral and striatal grafts to the lesioned striatum. TINS 10(12):509–516 4. Jonsson ME et al (2009) Identification of transplantable dopamine neuron precursors at different stages of midbrain neurogenesis. Exp Neurol 219(1):341–354 5. Carvey PM et al (2001) A clonal line of mesencephalic progenitor cells converted to dopamine neurons by hematopoietic cytokines: a source of cells for transplantation in Parkinson’s disease. Exp Neurol 171(1):98–108 6. Zeng X et al (2004) Dopaminergic differentiation of human embryonic stem cells. Stem Cells 22(6):925–940 7. Chiba S et al (2008) Noggin enhances dopamine neuron production from human embryonic stem cells and improves behavioral outcome after transplantation into Parkinsonian rats. Stem Cells 26(11):2810–2820 8. Hedlund E et al (2008) Embryonic stem cellderived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment by fluorescence-activated cell sorting and function in an animal model of Parkinson’s disease. Stem Cells 26(6):1526–1536 9. Kriks S et al (2011) Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480 (7378):547–551 10. Arenas E (2010) Towards stem cell replacement therapies for Parkinson’s disease. Biochem Biophys Res Commun 396(1):152–156 11. O’Keeffe FE et al (2008) Induction of A9 dopaminergic neurons from neural stem cells improves motor function in an animal model of Parkinson’s disease. Brain 131(Pt 3):630–641 12. Mendez I et al (2008) Dopamine neurons implanted into people with Parkinson’s disease survive without pathology for 14 years. Nat Med 14(5):507–509 13. Deierborg T et al (2008) Emerging restorative treatments for Parkinson’s disease. Prog Neurobiol 85(4):407–432 14. Redmond DE Jr et al (2007) Behavioral improvement in a primate Parkinson’s model is associated with multiple homeostatic effects of human neural stem cells. Proc Natl Acad Sci U S A 104(29):12175–12180

15. Sonntag KC et al (2007) Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin. Stem Cells 25(2):411–418 16. Ko JY et al (2007) Human embryonic stem cellderived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons. J Neurochem 103(4):1417–1429 17. Yasuhara T et al (2006) Transplantation of human neural stem cells exerts neuroprotection in a rat model of Parkinson’s disease. J Neurosci 26(48):12497–12511 18. Lindvall O, Kokaia Z (2006) Stem cells for the treatment of neurological disorders. Nature 441(7097):1094–1096 19. Mendez I et al (2005) Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson’s disease. Brain 128 (Pt 7):1498–1510 20. Correia AS et al (2005) Stem cell-based therapy for Parkinson’s disease. Ann Med 37 (7):487–498 21. Bjorklund A (2005) Cell therapy for Parkinson’s disease: problems and prospects. Novartis Found Symp 265:174–186 Discussion 187, 204–211 22. Roy NS et al (2006) Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomeraseimmortalized midbrain astrocytes. Nat Med 12(11):1259–1268 23. Armstrong RJ et al (2003) Transplantation of expanded neural precursor cells from the developing pig ventral mesencephalon in a rat model of Parkinson’s disease. Exp Brain Res 151 (2):204–217 24. Ben-Hur T et al (2004) Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats. Stem Cells 22(7):1246–1255 25. Borlongan CV, Sanberg PR (2002) Neural transplantation for treatment of Parkinson’s disease. Drug Discov Today 7(12):674–682 26. Daadi MM et al (2012) Dopaminergic neurons from midbrain-specified human embryonic stem cell-derived neural stem cells engrafted in a monkey model of Parkinson’s disease. PLoS One 7(7):e41120 27. Grealish S et al (2010) The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson’s disease. Brain 133(Pt 2):482–495

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28. Kim JH et al (2002) Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418(6893):50–56 29. Lee SH et al (2000) Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 18 (6):675–679 30. Kawasaki H et al (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28 (1):31–40 31. Perrier AL et al (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 101 (34):12543–12548 32. Hong S et al (2008) Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons. J Neurochem 104(2):316–324 33. Yan Y et al (2005) Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23 (6):781–790 34. Schulz TC et al (2004) Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. Stem Cells 22(7):1218–1238

35. Vazin T et al (2009) A novel combination of factors, termed SPIE, which promotes dopaminergic neuron differentiation from human embryonic stem cells. PLoS One 4(8):e6606 36. Hayashi H et al (2008) Meningeal cells induce dopaminergic neurons from embryonic stem cells. Eur J Neurosci 27(2):261–268 37. Cho MS et al (2008) Highly efficient and largescale generation of functional dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 105(9):3392–3397 38. Ko JY et al (2009) Conditions for tumor-free and dopamine neuron-enriched grafts after transplanting human ES cell-derived neural precursor cells. Mol Ther 17(10):1761–1770 39. Ueno M et al (2006) Neural conversion of ES cells by an inductive activity on human amniotic membrane matrix. Proc Natl Acad Sci U S A 103(25):9554–9559 40. Kim DW et al (2006) Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells. Stem Cells 24(3):557–567 41. Cai J et al (2009) The role of Lmx1a in the differentiation of human embryonic stem cells into midbrain dopamine neurons in culture and after transplantation into a Parkinson’s disease model. Stem Cells 27(1):220–229

Chapter 8 Midbrain Dopaminergic Neurons Differentiated from Human-Induced Pluripotent Stem Cells Fabiano Arau´jo Tofoli, Ana Teresa Silva Semeano, A´gatha Oliveira-Giacomelli, Maria Carolina Bittencourt Gonc¸alves, Merari F. R. Ferrari, Lygia Veiga Pereira, and Henning Ulrich Abstract The work with midbrain dopaminergic neurons (mDAN) differentiation might seem to be hard. There are about 40 different published protocols for mDAN differentiation, which are eventually modified according to the respective laboratory. In many cases, protocols are not fully described, failing to provide essential tips for researchers starting in the field. Considering that commercial kits produce low mDAN percentages (20–50%), we chose to follow a mix of four main protocols based on Kriks and colleagues’ protocol, from which the resulting mDAN were engrafted with success in three different animal models of Parkinson’s disease. We present a differential step-by-step methodology for generating mDAN directly from humaninduced pluripotent stem cells cultured with E8 medium on Geltrex, without culture on primary mouse embryonic fibroblasts prior to mDAN differentiation, and subsequent exposure of neurons to rock inhibitor during passages for improving cell viability. The protocol described here allows obtaining mDAN with phenotypical and functional characteristics suitable for in vitro modeling, cell transplantation, and drug screening. Key words Induced pluripotent stem cells, Cell reprogramming, Cell differentiation, Dopaminergic neurons, Neural differentiation, Dopaminergic differentiation, Neurodegenerative disease models, Parkinson’s disease, In vitro modeling

1

Introduction Ethical issues and difficult access to human tissue samples have resulted in the wide use of animal models for the elucidation of neurodegenerative diseases and possible treatments. However, improvements in research techniques enabled the application of more refined methods in the study of these pathologies, such as the use of human-induced pluripotent stem cells (hiPSC), which circumvent the limited similarity between animal and human models [1].

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_8, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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hiPSC are originated by reprogramming terminaldifferentiated cells, such as fibroblasts, lymphocytes and erythroblasts, into a pluripotent state. This was first achieved with a human cell line in 2007 by both Takahashi et al. [2] and Yu et al. [3], when the introduction of a combination of transcription factors into fibroblast cells resulted in pluripotent cells. Similar to human embryonic stem cells, these are capable of proliferating and differentiating into any cell type [2, 3]. Lately, the progress of reprogramming methods allowed the development of alternative protocols. The use of erythroblasts provides a more rapid and less invasive cell source for reprogramming, and the use of virus-free and integration-free approaches made hiPSC more suitable for cell therapy and genetic studies [4–6]. In vitro models, genetic analysis of patients, drug screening, and cell transplantation are some of the possible applications for hiPSC [7]. In these studies, differentiation into more specialized cell types is frequently applicable, and a variety of differentiation protocols emerged along with hiPSC establishment. The dopaminergic differentiation described herein derives from a protocol established by Kriks et al. [8] that was adapted and optimized [9] and results in midbrain dopaminergic neurons (mDAN) matching phenotypes found in substantia nigra, the main area affected in Parkinson’s disease (PD) patients. Zhang et al. [10] published a more detailed protocol for generating midbrain dopaminergic neurons (mDAN). Recently, Nolbrant et al. [11] suggested an upgraded version of the previous protocol, yielding increased percentages and reproducibility of mDAN differentiation. In parallel, Jo et al. [12] generated mDAN organoids in vitro, introducing a novel tool for the study of PD pathophysiology. The protocol described here focuses mainly on inhibition of SMAD signaling pathways induced by transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP). These are known to be involved in embryonic development and differentiation, proliferation and apoptosis, among other functions [8, 13–15]. In order to enrich mDAN populations, in our protocol we combined the use of some inhibitors in specific differentiation steps (Fig. 1): (a) SB431542, a Lefty/Activin/TGFβ inhibitor through ALK4, ALK5, and ALK7 receptor phosphorylation; (b) LDN193189, an inhibitor of ALK2 and ALK3 receptors and consequently of the BMP pathway; (c) CHIR99021, a selective GSK3-β inhibitor that promotes Wnt pathway activation; and (d) DAPT, which indirectly inhibits the Notch pathway by suppressing the γ-secretase complex [8, 14, 16–19]. Additionally, sonic Hedgehog (SHH) pathway is activated by the use of purmorphamine in combination with SHH in early differentiation stages. Our protocol is divided into three steps: first differentiating hiPSC into neural stem cells (NSC) during 11 days, followed by differentiation into neural progenitor cells (NPC) for additional 9 days, and finally

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Fig. 1 Modulatory reagents used and their target signaling pathways. Green bar, activation; red bar, inhibition; hiPSC induced pluripotent stem cell, NSC neural stem cell, NPC neural progenitor cell, mDAN midbrain dopaminergic neuron, BMP bone morphogenetic protein pathway, TGFβ1/3 transforming growth factor beta 1 and 3 pathway, SHH sonic hedgehog pathway, purm. purmorphamine, FGF-8 fibroblast growth factor 8 pathway, BDNF brain-derived neurotrophic factor, GDNF glial cell line-derived neurotrophic factor

into mDAN from the 20th day on. During days 25–90 mDAN are observed. Culture morphology throughout the culture protocol is presented in Fig. 2, including erroneous detaching cells. Dopaminergic differentiation protocols aim to produce functional cells, similar to those obtained by primary culture of midbrain dopaminergic neurons, either for transplantation or for obtaining a reliable in vitro model, optimizing the efficacy and safety of stem cell-based therapeutic approaches. Determination of cell-type-specific marker expression and gene expression with immunological assays and reverse transcriptase quantitative polymerase chain reaction, respectively, are needed to confirm whether the phenotype of hiPSC-derived cells resembles that of human dopaminergic neurons. During the first stages of neural differentiation, downregulation of OCT4 and SOX2 endogenous pluripotency gene expression simultaneously occurs together with the upregulation of neural progenitor NES and PAX6 gene expression [20–23]. Midbrain neuron specification requires the expression of FOXA2 and LMX1A transcription factors [24, 25]. One of the gold standard markers for identification of mDAN is tyrosine hydroxylase (TH), a rate-limiting enzyme involved in dopamine synthesis, converting tyrosine into L-dihydroxyphenylalanine (L-DOPA) and subsequently into dopamine by the action of DOPA decarboxylase. TH-positive cells also express molecular markers for immature neurons (TuJ1), mature neurons (MAP 2 and NeuN), and dopaminergic neurons (LMX1B, NURR1, PITX3, DAT, and VMAT) [26–29], with increasing intensity as differentiation proceeds. The

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Fig. 2 Cell morphology changes during the progress of differentiation into mDAN. (a–c) hiPSC culture submitted to the differentiation protocol exhibiting morphological changes between 0 and 36 days in vitro, with a concentration of approximately 2
105 cells/cm2. (d) Cells on the 36th day of differentiation at lower concentration. (e) Detaching cells between day 8 and 13 of in vitro differentiation

herein protocol generates neurons with staining patterns characteristic of midbrain dopaminergic neurons, as shown in Figs. 3 and 4. Complementary quantitative analyses of dopamine transporter (DAT) and intracellular dopamine were also performed in the hiPSC-derived neurons. Flow cytometry analysis showed that 70% of the cells obtained from differentiation positively marked intracellular dopamine and 54% also co-expressed DAT (data not shown). Neuronal cultures that include cells positive for both TH and G-protein-regulated inward-rectifier potassium channel 2 (GIRK2) expression are mDAN representative for the A9

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Fig. 3 Neural progenitor cells markers. Cells on the 11th day of differentiation expressing NPC markers, such as Nestin, Sox2, and FoxA2

population [30, 31], typical for the substantia nigra pars compacta. Previous publications describing differentiation of dopaminergic neurons from hiPSCs did not report differentiation efficiencies, with the exception of a detailed quantitative immunostaining study, where dopamine-producing and TH-positive neurons represented around 20% of the total cells and 50% of the neurons, in which about 17% of the population co-expressed GIRK2 [32]. Once the specific cell identity has been validated based on expression patterns of differentiation-relevant proteins and transcripts, it is quite relevant to ascertain the neuronal functionality of originated mDAN. The patch-clamp electrophysiology technique (Fig. 5a) allows investigating spontaneous firing of nerve impulses and action potentials with spike frequency adaptation after a depolarizing current injection. Stimulation-evoked catecholamine release can be measured by patch amperometry, by fast-scan cyclic voltammetry, or by high-performance liquid chromatography (HPLC). Neuronal functionality can also be investigated by detecting transient increases in free intracellular calcium concentration upon stimulation with neurotransmitters, such as dopamine. Mature functional neurons trigger action potentials when stimulated by current injection in the whole-cell patch clamp in the current-clamp configuration. The maturation of hiPSC-derived neurons can be monitored over time by evaluation of their electrophysiological properties by whole-cell configuration patch clamping in current-clamp configuration. With the protocol here presented, neurons in early stages of maturation (35 days) fire only a single action potential even during long depolarizing current steps (Fig. 5c); however, while differentiation culture proceeds (55 days), repetitive action potentials are observed upon stimulation (Fig. 5e) [32]. Spontaneous rhythmic action potentials also occur in mature cultures, with a continuous increase in firing frequencies during maturation. Gap-free current-clamp recordings of

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Fig. 4 Early midbrain dopaminergic neurons markers. Cells on the 37th day of differentiation expressing immature neuron marker Tuj1 (a) and mDAN markers, such as TH (b), Nurr1 (c), and PitX3 (d). Dopaminergic markers (TH and PitX3) co-localize with neuron marker (Tuj1) (e). mDAN midbrain dopaminergic neurons, DAPI 40 ,6-diamidino-2-phenylindole, TH tyrosine hydroxylase, Nurr1 nuclear receptor-related 1 protein, PitX3 pituitary homeobox 3, Tuj1 neuron-specific class III beta tubulin

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Fig. 5 Electrophysiological characterization of human hiPSC-derived mDAN. (a) Phase-contrast micrograph of neurons clamped by the borosilicate pipette, scale bar 40 μm. (b–e) Representative current-clamp recordings in whole-cell configuration of differentiated neuronal cultures. (b, d) Stimulation-induced action potential firing of neurons during current injection after 35 and 55 days of dopaminergic differentiation, respectively. (c) Tonic spontaneous action potentials after 35 and 55 days of dopaminergic differentiation, respectively

pace-making activity in mDAN after 35 and 55 days of differentiation are shown in Fig. 5d and 5e, respectively. Mature dopaminergic neurons obtained from this protocol acquired the maximum frequency of action potential firing, besides showing autonomic activity of slow 10 Hz rates, which are typically for mature mDAN with spikes from 8 to 15 Hz [33, 34]. Intracellular calcium waves and signaling play key roles in cellular physiology. For instance, the importance of L-type calcium channels has been documented in the cellular function of dopaminergic neurons from the substantia nigra pars compacta [35, 36]. Free intracellular calcium-transient responses upon stimulation with neurotransmitters serve as indicator for the progress of maturation and functionality of these mDAN, whose low amplitudes and slow transients become sharper and larger over time [32, 34]. Spontaneous intracellular calcium oscillations of neurons

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Fig. 6 Free intracellular calcium ([Ca2+]i) transients in human hiPSC-derived mDAN. (a) Representative curve of spontaneous oscillatory [Ca2+]i signals of mature neuronal cells. (b) Variation of free intracellular calcium concentration evoked by 10 μM dopamine injected in time 0

(after 55 days of differentiation) obtained from the protocol described herein can be seen in Fig. 6a. Additionally, an increased intracellular calcium response upon stimulation with dopamine is shown in Fig. 6b, supporting the presence and responsiveness of these receptors in cultured dopaminergic neurons. The protocol described generates mDAN with proven morphological, protein expression and physiological characteristics, suitable for in vitro modeling, cell transplantation and drug screening.

2

Materials 1. Accutase cell detachment solution—BD Biosciences, cat. no. 561527. 2. Ascorbic acid (AA)—Sigma-Aldrich, cat. no. 4544. 3. B27 supplement 50
, serum free—Thermo Fisher Scientific, cat. no. 17504-044. 4. Bovine serum albumin (BSA) 7.5%—Thermo Fisher Scientific, cat. no. 15260-037. 5. Brain-derived neurotrophic factor (BDNF)—Recombinant Human R&D Systems, cat. no. 248-BD-025. 6. CHIR 99021—Millipore, cat. no. 361571. 7. DAPT—Tocris, cat. no. 2634/10. 8. Dimethyl sulfoxide (DMSO) Hybri-Max—Sigma-Aldrich, cat. no. D2650. 9. Distilled water—Thermo Fisher Scientific, cat. no. 15230-170. 10. Dulbecco’s modified Eagle’s medium F12 (DMEM F12)— Thermo Fisher Scientific, cat. no. 11320-033.

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11. Dulbecco’s phosphate-buffered saline (dPBS)—Thermo Fisher Scientific, cat. no. 14190-144. 12. E8 medium—Thermo Fisher Scientific, cat. no. A15169-01. 13. Fetal bovine serum, embryonic stem cell qualified (FBS ESQ)—Thermo Fisher Scientific, cat. no. 16141-079. 14. Fibroblast growth factor 8b (FGF-8b) Recombinant Human/ Mouse Protein—R&D Systems, cat. no. 423-F8-025. 15. Geltrex (Gtx)—Thermo Fisher Scientific, cat. no. A14133-02. 16. Glial cell line-derived neurotrophic factor (GDNF) Recombinant Human—R&D Systems, cat. no. 212-GD-050. 17. Glutamax—Thermo Fisher Scientific, cat. no. 35050-061. 18. Human serum albumin (HSA)—Irwine, cat. no. 9988. 19. Knockout DMEM (KO DMEM)—Thermo Fisher Scientific, cat. no. 10829-018. 20. Knockout serum replacement (KSR)—Thermo Fisher Scientific, cat. no. 10828. 21. LDN 193189—Stem Macs, cat. no. 130-103-925. 22. N2 supplement no. 17502-048.

100
—Thermo

Fisher

Scientific,

cat.

23. N6,20 -O-Dibutyryladenosine 30 ,50 -cyclic monophosphate sodium salt (cAMP)—Sigma, cat. no. D0260/100MG. 24. Neurobasal medium—Thermo Fisher Scientific, cat. no. 21103049. 25. Nonessential amino acid, minimum essential medium (MEM NEAA)—Thermo Fisher Scientific, cat. no. 11140-050. 26. Penicillin/streptomycin (Pen/Strep)—Thermo Fisher Scientific, cat. no. 15140-122. 27. Purmorphamine (Purm)—StemGent, cat. no. 04-0009. 28. SB431542—Sigma, cat. no. S4317/Tocris Bioscience, cat. no. 1614. 29. Sonic Hedgehog (SHH) C25II Recombinant Mouse—R&D Systems, cat. no. 464-SH-200. 30. Transforming growth factor β3 (TGFβ3) Recombinant Human Protein—R&D Systems, cat. no. 243-B3-002. 31. TrypLE—Thermo Fisher Scientific, cat. no. 12604-013. 32. Versene—Thermo Fisher Scientific, cat. no. 15040-066. 33. Water for embryo transfer, sterile-filtered, BioXtra—SigmaAldrich, cat. no. W1503-100ML. 34. Y27632 dihydrochloride—Sigma-Aldrich, cat. no. SCM075. 35. β-Mercaptoethanol 1000
—Thermo Fisher Scientific, cat. no. 21985.

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Methods

3.1 Media, Supplements, and Matrix Preparation

Supplements were reconstituted according to manufacturers’ instructions at final concentrations described in Table 1 (see Note 1). Other dilutions and recommendations can be found in [9–11]. Once growth factors and inhibitors have been thawed, do not freeze them again (be sure to make aliquots in small volumes). It is strongly recommended to freshly add supplements to the medium. First, aliquot medium to final volumes used in your experiments to avoid warming up unnecessary quantities. Warm medium to 37  C and put in supplements right before adding to the cell culture (see Notes 2 and 3). To prepare culture media, combine reagents as indicated in Table 2. Ready-to-use media must be kept at 4  C. For the matrix preparation, aliquots of Gtx 1:100 in a volume of 100 μL are recommended. To avoid gelatinization, all Gtx manipulation must be done on ice. Prepare 1
Gtx by adding one aliquot (100 μL) in 10 mL of cold DMEM in culture biosafety cabinet. Add the appropriate volume (see Table 3) in a 6-well or 12-well plate according to protocol stage and incubate for 30 min at 37  C, 1 h at room temperature, or 24 h at 4  C (see Notes 4 and 5). Table 1 Final concentration of reagents and medium supplements Reagent

Final concentration

cAMP

250 mM

AA

200 μM

BDNF

20 ng/mL

CHIR 99021

3 μM

DAPT

10 μM

FGF8

100 ng/mL

GDNF

20 ng/mL

LDN 193189

100 nM

Purm

2 μM

ROCK inhibitor

10 μM

SB 431542

10 μM

SHH C25 II

100 ng/mL

TGFβ3

1 ng/mL

cAMP cyclic adenosine monophosphate, AA ascorbic acid, BDNF brain-derived neurotrophic factor, FGF-8 fibroblast growth factor 8, GDNF glial cell line-derived neurotrophic factor, Purm purmorphamine, SHH sonic hedgehog, TGFβ3 transforming growth factor beta 3

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Table 2 KSR, N2, and B27 medium compositions KSR medium composition

Volume (mL)

N2 medium composition

Volume (mL)

B27 medium composition

Volume (mL)

DMEM KO

8.2

DMEM F12

9.7

Neurobasal

9.6

MEM NEAA 100


0.1

Pen/Strep 100


0.1

Pen/Strep 100


0.1

Pen/Strep 100


0.1

Glutamax 100


0.1

Glutamax 100


0.1

Glutamax 100


0.1

Filter

β-Mercaptoethanol 1000


0.01

N2 supplement 100


Filter

Filter

Final volume

KSR 15%

0.1 10

B27 supplement 50
Final volume

0.2 10

1.5

Final volume

10

DMEM Dulbecco’s modified Eagle’s medium, KO knockout, MEM NEAA minimum essential medium nonessential amino acids, Pen/Strep penicillin-streptomycin

Table 3 Gtx 1
concentrated volume according to plate type Plate

Surface area (cm2)

Gtx volume (μL) for 1
Gtx

Gtx volume (μL) for 2
Gtx

6 wells

9.5

1250

2500

12 wells

3.8

500

1000

24 wells

1.9

250

500

48 wells

0.95

125

150

96 wells

0.34

45

90

3.2

hiPSC Culture

See Notes 6 and 7. 1. Unfreeze cryopreserved hiPSC into FBS ESQ 90% + DMSO 10% by transferring them into a 15 mL centrifuge tube and diluting in 5 mL of E8 medium +10 μM rock inhibitor [37–39] (see Note 8). 2. Centrifuge for 3 min at 200
g and discard the supernatant without disturbing the pellet. 3. Add 1 mL E8 medium +10 μM ROCK inhibitor and gently resuspend the pellet through up and down movements until there is no visible pellet (must have small clumps). 4. Discard Gtx excess from a 6-well plate previously treated with 1
Gtx, add 2 mL E8 medium + rock inhibitor per well, and seed 500 μL, 300 μL, and 200 μL of resuspended hiPSC in

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3 wells containing 2 mL of E8 medium with 10 μM ROCK inhibitor per well (see Note 9). 5. After 24 h, change culture medium to E8 medium without ROCK inhibitor. Choose the well with less differentiated hiPSC and with about 30–50% confluence, maintaining them in culture until achieving 80–90% confluence. Manually remove differentiated colonies from the plate with a 200 μL tip (see Note 10). 6. Dissociate hiPSC culture with dissociation agent until single cells are observed (see Note 11). Count cells using a hemocytometer. 7. Seed 2.5 to 3.5
104 cells/cm2 diluted in E8 medium with 10 μM ROCK inhibitor. 8. After 24 h, change 100% culture medium to E8 medium without ROCK inhibitor. 9. Change E8 medium every day until cells are 90–100% confluent. hiPSC growth rates vary according to lineages (see Note 12). 3.3 Dopaminergic Differentiation

1. Discard medium and wash one time with dPBS. 2. Change cell medium as specified in Table 4, considering this first exchange as day 0 (Fig. 7) (see Notes 13 and 14). 3. After day 21, change medium every 48 h until desired mDAN maturation is obtained (the here presented mDAN were characterized after 30 days of differentiation).

3.4 Neural Cell Passage

In the standard protocol, neural cell passage is made only at the 20th day of differentiation. Also there are two variations with one additional passage on the 11th or 13th day of differentiation (see Notes 16 and 22). 1. Add 10 μM ROCK inhibitor to the well and incubate at 37  C during 30 min. 2. Discard medium, add 1 mL of pre-warmed accutase (37  C) with 10 μM rock inhibitor, and incubate at 37  C for 10 min. 3. Gently resuspend cells with around six up and down movements, avoiding air bubbles, and incubate at 37  C for 10 min. Check in the microscope and repeat the process until single cells are observed or reach the maximum of six repetitions (see Note 23). 4. Add 2 mL of B27 medium with 10 μM ROCK inhibitor to inactivate accutase. 5. Slowly transfer sample to a 15 mL centrifuge tube by dropping it in the center of the tube (do not flow through the border) and centrifuge for 5 min at 200
g.

KSR

KSR

KSR

KSR

KSR 75%: N2 25%

KSR 75%: N2 25%

KSR 50%: N2 50%

KSR 50%: N2 50%

KSR 25%: N2 75%

KSR 25%: N2 75%

B27

B27

B27

B27

B27

B27

B27

B27

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

LDN + SB + SHH + Pur + FGF8 + CHIR

LDN + SB + SHH + Pur + FGF8

LDN + SB + SHH + Pur + FGF8

3 mL

3 mL

BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

2.5 mL BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

2.5 mL BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

2.5 mL BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

3 mL

Stop with SHH, Pur, FGF8

Stop with SB

See Notes 15 and 16

hiPSC 80–95% confluence

Observation

(continued)

Optional passage: See Note 17 to choose best passage day

BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA + CHIR Optional passage: See Note 17 to choose best passage day See Notes 18 and 19

LDN + CHIR

LDN + CHIR

LDN + CHIR

2.5 mL BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA + CHIR

2 mL

2 mL

2 mL

2 mL

1.5 mL LDN + CHIR

1.5 mL LDN + SHH + Pur + FGF8 + CHIR

1.5 mL LDN + SHH + Pur + FGF8 + CHIR

1.5 mL LDN + SB + SHH + Pur + FGF8 + CHIR

1 mL

1 mL

1 mL

1 mL

KSR

0

LDN + SB

Volume Supplements

Day Medium

Table 4 Medium composition throughout the timeframe protocol

Midbrain Dopaminergic Neurons Differentiated from hiPSC 109

1 mL

B27 + 10 μM ROCK inhibitor

B27

No change

B27

No change

B27

20

21

22

23

24

25

BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

See Note 21

See Note 21

Be sure to discard all medium to take out dead cells

Optional passage: See Note 17 to choose best passage day See Note 20

Observation

LDN LDN193189, SB SB431542, SHH sonic hedgehog, Pur purmorphamine, FGF-8 fibroblast growth factor 8, CHIR CHIR99021, BDNF brain-derived neurotrophic factor, GDNF glial cell line-derived neurotrophic factor, TGFβ3 transforming growth factor beta 3, cAMP cyclic adenosine monophosphate, AA ascorbic acid

1 mL

1 mL

1 mL

3 mL

B27

19

BDNF + GDNF + TGFβ3 + DAPT + cAMP + AA

Volume Supplements

Day Medium

Table 4 (continued)

110 Fabiano Arau´jo Tofoli et al.

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Fig. 7 Differentiation protocol overview. Neural differentiation (gray arrows on the top) occurs with medium change along the days of culture (green to orange transition, bottom). Blue bars represent supplements added to the medium. In the presented protocol, passage is made on the 20th day, although variations are possible as previously discussed. CHIR CHIR99021, FGF-8 fibroblast growth factor 8, Pur. purmorphamine, SHH sonic hedgehog, SB SB431542, LDN LDN193189, BDNF brain-derived neurotrophic factor, GDNF glial cell linederived neurotrophic factor, TGFβ3 transforming growth factor beta 3, cAMP cyclic adenosine monophosphate Table 5 Final volume of medium + supplements per well during passages in plates of different sizes Plate

Surface area (cm2)

Passage volume (μL/well)

6 wells

9.5

2500

12 wells

3.8

1000

24 wells

1.9

500

48 wells

0.95

250

96 wells

0.34

90

Volume suggested for 2
10 seeded cells per cm 5

2

6. Discard supernatant and add 2 mL B27 medium with 10 μM ROCK inhibitor and supplements per tube. Gently homogenize avoiding air bubbles. 7. For the passage on the 20th day of differentiation, dilute and seed 2
105 cells/cm2 in a 12-well plate, previously treated with 2
Gtx (Table 3). For passage on days 11 or 13, seed one sample per well in a 12-well plate, previously treated with 1
Gtx (cell counting is not necessary). For other plate configurations, see Table 5 (also see Notes 24–26). 8. Gently shake the plate in all directions and incubate at 37  C, 5% CO2.

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Notes 1. BSA can be changed to HSA for xeno-free composition. 2. KSR, N2, and B27 supplements must be thawed under cold temperature (ice or 4  C; never warm up at room temperature or 37  C). Small aliquots are highly recommended and must be stored at 20  C. Avoid freeze-thaw cycles. 3. Although the majority of protocols uses hiPSC culture on Matrigel with mTeSR1 or embryonic stem cell (ESC)conditioned medium before starting mDAN differentiation, we did not test mDAN differentiation with hiPSC cultured on mouse embryonic fibroblast (MEF) with conditioned medium, since some authors pointed that commercial medium (E8 or mTeSR1) could induce hiPSC to differentiate into specific cell types. We started testing hiPSC culture on 1
Gtx with (a) mTeSR1, (b) 50% mTeSR1 + 50% E8 medium, and (c) E8 medium. After mDAN differentiation, our results showed that neurons obtained by procedure a. or b. detached more easily from the matrix after 25 days than hiPSC did which had been only cultured with E8 medium. We hypothesize that E8 medium maintains hiPSC in a more undifferentiated state, since we were able to reprogram somatic cells into hiPSC using E8 but not mTeSR1 medium [40–42]. 4. Carefully follow manufacturer’s specification when working with Gtx matrix. See protocol of Tomishima [15] for tips or follow same instructions for Matrigel as described by Zhang et al. [10]. 5. We tested low concentrations (1 and 2 μg/mL) of laminin as matrix, which resulted in increased cell detachment. Although higher concentrations (3, 4, and 5 μg/mL) decreased detachment, cells tended to migrate and accumulate in the center of the well. Different concentrations of combined polyornithine/ laminin/fibronectin matrix promoted high cell detachment. 6. Do not replicate or cryopreserve hiPSC cultures when confluence has passed 90%. 7. When hiPSC reprogramming protocol involves decondensation of chromatin (e.g., by sodium butyrate or valproic acid), to improve cell reprogramming by plasmids, we strongly recommend to make few passages without them before starting the differentiation. These compounds might compromise hiPSC differentiation. 8. Initial hiPSC concentration seems irrelevant, since neural differentiation will start when cultures have achieved 80–95% of confluence.

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9. For immunocytochemistry of hiPSC differentiated into immature neurons, seed hiPSC into 96-well plates. 10. If hiPSC colonies do not present the characteristic morphologies (see [43]), remove cells with a sterile 200 μL micropipette tip and visualize them under a microscope/magnifier under sterile conditions and subject them to fragment passage using Versene. If the expected morphology is observed and cells have reached 90% of confluence, split cells from 1 well into 3 or 4 wells (6-well plate). Treat hiPSC culture with dissociation agent (Versene) for 3–5 min at 37  C. Discard Versene before cells start to detach from the matrix, add E8 medium +10 μM rock inhibitor, and incubate for 3 min. Dissociate hiPSC colonies using a 200 μL micropipette tip in hash movements. Transfer cell suspensions into 50 mL tube and centrifuge for 3 min at 200
g and room temperature. Collect supernatant and add the final volume of E8 medium + rock inhibitor to a final concentration of 10 μM (number of desired wells x 2 mL). Make slow up and down movements with a micropipette, until no pellet is seen (must have small clumps), and transfer 2 mL/ well into 6-well plates. Gently shake the plate and incubate at 37  C and 5% CO2 atmosphere in a cell culture incubator. Change E8 medium every day. 11. Versene, ReLSR (for fragment passage), accutase, and TrypLE enzyme (for single-cell passage) were tested for cell dissociation, giving all similar results. Independent of the chosen one, make sure that there are no hiPSC clumps before proceeding to next steps. 12. For more hiPSC handling procedures, we advise the “Protocol for the use of hiPCS,” from the European Bank for induced pluripotent Stem Cells (EBiSC) or guides for human ESC and hiPSC procedures from Thermo Fisher, R&D Systems, or stem cell manufacturers. 13. Do not drop medium directly on cell monolayers. Add medium slowly by flowing through the border. 14. Medium volumes are adjusted to 1 well of a 12-well plate (3.8 cm2). If necessary, adjust volume according to plate size. 15. There are variants of SHH. Tomishima [15] mentioned the use of SHH (461-SH); however, the isoleucine-modified version SHH 25CII (464-SH) was chosen due to better results. Nolbrant et al. [11] strongly recommend the use of SHH24 for improving dopaminergic neurons differentiation. Although Zhang et al. [10] successfully changed SHH to smoothened agonist (SAG), we failed to establish this protocol due to increased cell detachment. 16. As hiPSC lineages have different characteristics, the best choice of passage numbers and differentiation stages may vary. Lorenz

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Studer’s group described three variations: (a) a single passage on the 20th day [8], (b) one passage on the 11th day and one on the 20th day [14], and (c) one passage on the 13th day and one on the 20th day of differentiation [9]. We recommend to start with variation (a) if cells start to detach between 9th and 13th days, test variation (b) and then (c). We observed that, although all variations worked in hiPSC control, neural differentiation with other hiPSC lineages could present poor cell number on the 20th day using variation c. 17. Although the majority of protocols uses cAMP at 500 μM [8, 9, 11, 12]. Li et al. [44] use higher concentration (1 mM), while Zhang et al. [10] use lower concentration (100 μM). We tested 500, 250, and 150 μM, obtained similar results, and chose the concentration of 250 μM for our differentiation assays [12, 45]. 18. We tested two L-ascorbic acid compounds from Sigma (catalog numbers A5960 and A4544). We observed less neuron detachment following passaging using Sigma A4544. 19. To minimize or prevent mDAN detachment, some groups add laminin 1 μg/mL to the B27 medium [12, 45–48]. 20. For preventing medium acidification and DNA damage due to the increasing confluence [49], increase medium volume by 0.5-fold the initial volume (according to the chosen well configuration) every 4 days (see Table 4). 21. To prevent cell detachment during medium change after day 21 of differentiation, keep 10% of the old medium and add fresh medium up to the final volume expected. 22. The mDAN concentration to be chosen on the 20th day passage is controversial. Some protocols use higher concentrations, such as 8
105 cells/cm2, while others use lower concentration, as 1
105 cells/cm2 [8–12, 14, 15, 17, 45–48]. We tested 0.25, 0.5, 0.75, 1, 1.5, 2, and 4.5
105 cells/cm2. The minimal concentration eligible for mDAN differentiation was 1
105 cells/cm2 (e.g., for patch clamp assays), while 2
105 cells/cm2 presented optimal results. Certain proximity between cells is required for a better differentiation, since neurite formation and cell-to-cell communication are essential for neuron survival and homeostasis maintenance. The concentration of 4.5
105 cells/cm2 originated cells with decreased expression of mDAN markers. 23. We observed that the use of a strainer or vigorous up and down movements to dissociate clumps drastically decrease cell viability. Avoid more than a total of 60 min exposure to a dissociation agent during this step.

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24. 12-well plates are generally used for neural differentiation. After passage on the 20th day of differentiation, seed cells according to the experiment that will be subsequently performed. For example, for flow cytometry and RNA extraction, differentiation in 12-well plate is advised. For patch-clamp assay, 24-well plates are more suitable, and 96-well plates are sufficient for immunocytochemistry. 25. Neurons dissociated into single cells tend to regroup within minutes, forming clumps. After counting, do not vigorously enforce dissociation in order to avoid cell death. Pipet up and down slowly to minimize clumps and transfer into wells. 26. We observed that the usage of 2
Gtx on the 20th day passage decreases cell detachment, increases cell confluency, and presents more prominent fluorescence in immunocytochemistry assay when compared to 1
Gtx. Concentrations higher than 2
Gtx increased differentiation into other neuronal cell types.

Acknowledgments LVP is grateful for grant support by Fundac¸˜ao de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP -CEPID 13/08135-2), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico/ Departamento de Cieˆncia e Tecnologia do Ministe´rio da Sau´de (CNPq/MS/DECIT- 24/2014), Banco Nacional de Desenvolvimento Econoˆmico e Social (BNDES), Financiadora de Estudos e Projetos (FINEP), and 2010 Gaucher Generation grant program by Genzyme Corporation. HU is grateful for grant support by FAPESP (Project No. 2012/50880-4) and CNPq (Project No. 306429/2013-6). MFRF was awarded with research grants from FAPESP (2013/08028-1and 2015/18961-2) and CNPq (471999/2013-0 and 401670/2013-9). FTA (Project No. 2014/25487-3) is grateful for a doctorate fellowship granted by FAPESP. ATSS (Project No. 163310/2014-9), AOG (Project No. 141979/2014-3), and MCBG (Project No. 870458/1997-3) are grateful for a doctorate fellowship granted by CNPq. This study was financed in part by the Coordenac¸˜ao de Aperfeic¸oamento de Pessoal de Nı´vel Superior - Brazil (CAPES) - Finance Code 001. Special thanks to Mark Tomishima and Faria Zafar for online advising during the establishment of this protocol. References 1. Singh VK, Kalsan M, Kumar N, Saini A, Chandra R (2015) Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev

Biol 3:2. https://doi.org/10.3389/fcell. 2015.00002 2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K et al (2007) Induction of pluripotent stem cells from adult human

116

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fibroblasts by defined factors. Cell 131:861–872. https://doi.org/10.1016/j. cell.2007.11.019 3. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S et al (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920. https://doi.org/10.1126/ science.1151526 4. Chou B-K, Gu H, Gao Y, Dowey SN, Wang Y, Shi J et al (2015) A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xenofree culture conditions: a clinically compliant approach. Stem Cells Transl Med 4:320–332. https://doi.org/10.5966/sctm.2014-0214 5. Chou B-K, Mali P, Huang X, Ye Z, Dowey SN, Resar LM et al (2011) Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Res 21:518–529. https://doi.org/10.1038/cr.2011.12 6. Dowey SN, Huang X, Chou B-K, Ye Z, Cheng L (2012) Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression. Nat Protoc 7:2013–2021. https://doi.org/10.1038/nprot.2012.121 ´ , Ulrich H (2016) Purinergic recep7. Oliveira A tors in embryonic and adult neurogenesis. Neuropharmacology 104:272–281. https:// doi.org/10.1016/J.NEUROPHARM.2015. 10.008 8. Kriks S, Shim J-W, Piao J, Ganat YM, Wakeman DR, Xie Z et al (2011) Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480:547. https://doi.org/10.1038/ nature10648 9. Tomishima M (2012) Midbrain dopamine neurons from hESCs. Harvard Stem Cell Institute, Cambridge, MA. https://doi.org/10. 3824/stembook.1.70.1 http://www. stembook.org 10. Zhang P, Xia N, Reijo Pera RA (2014) Directed dopaminergic neuron differentiation from human pluripotent stem cells. J Vis Exp 91:51737. https://doi.org/10.3791/51737 11. Nolbrant S, Heuer A, Parmar M, Kirkeby A (2017) Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nat Protoc 12:1962–1979. https:// doi.org/10.1038/nprot.2017.078 12. Jo J, Xiao Y, Sun AX, Cukuroglu E, Tran H-D, Go¨ke J et al (2016) Midbrain-like organoids from human pluripotent stem cells contain

functional dopaminergic and neuromelaninproducing neurons. Cell Stem Cell 19:248–257. https://doi.org/10.1016/j. stem.2016.07.005 13. Boyer LF, Campbell B, Larkin S, Mu Y, Gage FH, Boyer LF et al (2012) Dopaminergic differentiation of human pluripotent cells. Curr Protoc Stem Cell Biol. John Wiley & Sons, Inc., Hoboken, NJ, pp 1H.6.1–1H.6.11. https://doi.org/10.1002/9780470151808. sc01h06s22 14. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275–280. https://doi.org/10.1038/nbt.1529 15. Tomishima M (2014) Neural induction – Dual SMAD inhibition. StemBook. http:// coredinates.org/resources/DSi.pdf. Accessed 10 Jan 2018 16. Fasano CA, Chambers SM, Lee G, Tomishima MJ, Studer L (2010) Efficient derivation of functional floor plate tissue from human embryonic stem cells. Cell Stem Cell 6:336–347. https://doi.org/10.1016/j.stem. 2010.03.001 17. Lee G, Chambers SM, Tomishima MJ, Studer L (2010) Derivation of neural crest cells from human pluripotent stem cells. Nat Protoc 5:688–701. https://doi.org/10.1038/nprot. 2010.35 18. Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N et al (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 101:12543–12548. https://doi.org/10. 1073/pnas.0404700101 19. Sundberg M, Bogetofte H, Lawson T, Jansson J, Smith G, Astradsson A et al (2013) Improved cell therapy protocols for Parkinson’s disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons. Stem Cells 31:1548–1562. https://doi.org/ 10.1002/stem.1415 20. Gerrard L, Rodgers L, Cui W (2005) Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cells 23:1234–1241. https://doi.org/10. 1634/stemcells.2005-0110 21. Shi G, Jin Y (2010) Role of Oct4 in maintaining and regaining stem cell pluripotency. Stem Cell Res Ther 1:1–9. https://doi.org/10. 1186/scrt39

Midbrain Dopaminergic Neurons Differentiated from hiPSC 22. Wu JQ, Habegger L, Noisa P, Szekely A, Qiu C, Hutchison S et al (2010) Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing. Proc Natl Acad Sci U S A 107:5254–5259. https://doi. org/10.1073/pnas.0914114107 23. Zhang S (2014) Sox2, a key factor in the regulation of pluripotency and neural differentiation. World J Stem Cells 6:305. https://doi. org/10.4252/wjsc.v6.i3.305 24. Friling S, Andersson E, Thompson LH, Jonsson ME, Hebsgaard JB, Nanou E et al (2009) Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells. Proc Natl Acad Sci 106:7613–7618. https://doi.org/10.1073/ pnas.0902396106 25. Lin W, Metzakopian E, Mavromatakis YE, Gao N, Balaskas N, Sasaki H et al (2009) Foxa1 and Foxa2 function both upstream of and cooperatively with Lmx1a and Lmx1b in a feedforward loop promoting mesodiencephalic dopaminergic neuron development. Dev Biol 333:386–396. https://doi.org/10. 1016/j.ydbio.2009.07.006 26. Arenas E, Denham M, Villaescusa JC (2015) How to make a midbrain dopaminergic neuron. Development 142:1918–1936. https:// doi.org/10.1242/dev.097394 27. Glaser T, Correˆa-Velloso J, Oliveira-Giacomelli ´ , Teng YD, Ulrich H (2017) Dopaminergic A and GABAergic neuron in vitro differentiation from embryonic stem cells. Humana Press, New York, NY, pp 45–53. https://doi.org/ 10.1007/978-1-4939-7024-7_3 28. Maxwell SL, Li M (2005) Midbrain dopaminergic development in vivo and in vitro from embryonic stem cells. J Anat 207:209–218. https://doi.org/10.1111/j.1469-7580.2005. 00453.x 29. Walle´n A, Perlmann T (2003) Transcriptional control of dopamine neuron development. Ann N Y Acad Sci 991:48–60. https://doi. org/10.1111/j.1749-6632.2003.tb07462.x 30. Chung CY, Seo H, Sonntag KC, Brooks A, Lin L, Isacson O (2005) Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum Mol Genet 14:1709–1725. https://doi.org/10.1093/ hmg/ddi178 31. Reyes S, Fu Y, Double K, Thompson L, Kirik D, Paxinos G et al (2012) GIRK2 expression in dopamine neurons of the substantia nigra and ventral tegmental area. J Comp Neurol 520:2591–2607. https://doi.org/10. 1002/cne.23051

117

32. Hartfield EM, Yamasaki-Mann M, Ribeiro Fernandes HJ, Vowles J, James WS, Cowley SA et al (2014) Physiological characterisation of human iPS-derived dopaminergic neurons. PLoS One 9:e87388. https://doi.org/10. 1371/journal.pone.0087388 33. Grace AA, Bunney BS (1984) The control of firing pattern in nigral dopamine neurons: single spike firing. J Neurosci 4:2866–2876 34. Grow DA, Simmons DV, Gomez JA, Wanat MJ, McCarrey JR, Paladini CA et al (2016) Differentiation and characterization of dopaminergic neurons from baboon induced pluripotent stem cells. Stem Cells Transl Med 5:1133–1144. https://doi.org/10.5966/ sctm.2015-0073 35. Guzman JN, Sanchez-Padilla J, Chan CS, Surmeier DJ (2009) Robust pacemaking in substantia nigra dopaminergic neurons. J Neurosci 29:11011–11019. https://doi.org/10.1523/ JNEUROSCI.2519-09.2009 36. Surmeier DJ (2007) Personal view calcium, ageing, and neuronal vulnerability in Parkinson’s disease. Neurology 6(10):933–938 37. Claassen DA, Desler MM, Rizzino A (2009) ROCK inhibition enhances the recovery and growth of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. Mol Reprod Dev 76:722–732. https://doi.org/10.1002/mrd.21021 38. Li X, Krawetz R, Liu S, Meng G, Rancourt DE (2008) ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum Reprod 24:580–589. https://doi.org/10.1093/ humrep/den404 39. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T et al (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25:681–686. https://doi.org/10. 1038/nbt1310 40. Lu HF, Chai C, Lim TC, Leong MF, Lim JK, Gao S et al (2014) A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells. Biomaterials 35:2816–2826. https://doi.org/10.1016/j.biomaterials. 2013.12.050 41. Tofoli FA, Dasso M, Morato-Marques M, Nunes K, Pereira LA, da Silva GS et al (2016) Increasing the genetic admixture of available lines of human pluripotent stem cells. Sci Rep 6:34699. https://doi.org/10.1038/ srep34699

118

Fabiano Arau´jo Tofoli et al.

42. Wang Y, Chou B-K, Dowey S, He C, Gerecht S, Cheng L (2013) Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions. Stem Cell Res 11:1103–1116. https://doi.org/10. 1016/j.scr.2013.07.011 43. Maddah M, Shoukat-Mumtaz U, Nassirpour S, Loewke K (2014) A system for automated, noninvasive, morphology-based evaluation of induced pluripotent stem cell cultures. J Lab Autom 19:454–460. https://doi.org/10. 1177/2211068214537258 44. Li M, Zou Y, Lu Q, Tang N, Heng A, Islam I et al (2016) Efficient derivation of dopaminergic neurons from SOX1 floor plate cells under defined culture conditions. J Biomed Sci 23:34. https://doi.org/10.1186/s12929016-0251-6 45. Zhang X-Q, Zhang S-C (2010) Differentiation of neural precursors and dopaminergic neurons from human embryonic stem cells. Methods Mol Biol 584:355–366. https://doi.org/10. 1007/978-1-60761-369-5_19

46. Lu J, Zhong X, Liu H, Hao L, Huang CT-L, Sherafat MA et al (2015) Generation of serotonin neurons from human pluripotent stem cells. Nat Biotechnol 34:89–94. https://doi. org/10.1038/nbt.3435 47. Qi Y, Zhang X-J, Renier N, Wu Z, Atkin T, Sun Z et al (2017) Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells. Nat Biotechnol 35:154–163. https://doi. org/10.1038/nbt.3777 48. Schuele B, Zafar F, Shin S, Michael D, Nguyen A, Flierl A, et al. (2016) Highefficiency differentiation into functional dopaminergic neurons from Parkinson’s patientsderived induced pluripotent stem cells. F1000Research. 5. https://doi.org/10.7490/ F1000RESEARCH.1112457.1 49. Jacobs K, Zambelli F, Mertzanidou A, Smolders I, Geens M, Nguyen HT et al (2016) Higher-density culture in human embryonic stem cells results in DNA damage and genome instability. Stem Cell Rep 6:330–341. https://doi.org/10.1016/j. stemcr.2016.01.015

Chapter 9 Generating Neural Stem Cells from iPSCs with Dopaminergic Neurons Reporter Gene Hyenjong Hong and Marcel M. Daadi Abstract Genetic reporters offer attractive approaches to investigate gene expression, gene function, and spatiotemporal assessment in vitro and in vivo. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of the dopamine neurotransmitter, and thus it has been used as a reliable marker for dopaminergic neurons in vitro and in vivo. Herein we describe a method for making iPSC lines with TH-green fluorescent protein reporter gene using CRISPR/Cas9 technique. Key words Tyrosine hydroxylase, Reporter, CRISPR/Cas9, Human pluripotent stem cells, Neural stem cell, Parkinson’s disease

1

Introduction Dopaminergic (DA) neurons are involved in many critical functions within the central nervous system (CNS), and dopamine neurotransmission impairment underlies a wide range of disorders from motor control deficiencies, such as Parkinson’s disease (PD) [1], to psychiatric disorders, such as alcoholism, drug addictions, bipolar disorders, and depression. Neural stem cell-based technology has potential to play an important role in developing efficacious biological and small molecule therapeutic products for dopamine dysregulation disorders. Tracking DA neurons with a reporter gene in real-time live cell culture system or brain tissue is an attractive and sound approach to study their development, physiology, and pathophysiology. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of dopamine. Thus, it has been used as a reliable marker for DA neurons [2]. Induced pluripotent stem cells (iPSCs) are generated from adult somatic cells through the introduction of several transcription factors [3, 4]. IPSCs may be used to derive patient’s own cells and model diseases, such as Parkinson’s disease in vitro [5, 6]. The combination of iPSC-based in vitro models and the TH gene

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_9, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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reporter approach will enable reliable systems for investigating dopaminergic neurons derived from healthy controls or patients with disease. Herein, we describe a method for establishing TH-derived green fluorescent protein-expressing reporter in human iPSCs using CRISPR/Cas9 technique [7–11]

2

Materials

2.1 Construction of gRNA Expressing Vector and Donor Vector

1. pSpCas9(BB)-2A-GFP (pX458) (Addgene). 2. AAVS1 hPGK-PuroR-pA donor (Addgene) [8]. 3. 5 M NaCl. 4. Polymerase chain reaction (PCR) machine. 5. TE buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 7.0 BbsI (New England BioLabs). 6. QIAquick Gel Extraction Kit (QIAGEN). 7. T4 ligase and buffer (New England BioLabs). 8. Gibson Assembly Master Mix (New England BioLabs). 9. One Shot Stbl3 Chemically Competent E. coli (Invitrogen).

2.2 Transfection of Plasmid into HEK293FT Cells and Verification of gRNA Using T7 Endonuclease 1

PureLink Quick Plasmid Miniprep Kit (Invitrogen). HEK 293FT cells (Life Technologies). DPBS, no calcium, no magnesium (Gibco). 0.05% Trypsin-EDTA (Gibco). 10% FBS media: Prepare DMEM, high glucose (Gibco) by supplementing 10%. HyClone FetalClone II Serum (GE Healthcare Life Sciences). Store at 4
C. Corning 60 mm TC-Treated Culture Dish (Corning). Opti-MEM, Reduced Serum Medium (Gibco). Lipofectamine 2000 transfection reagent (Life Technologies). T7 Endonuclease 1 (T7E1, New England BioLabs). Lysis buffer for genomic DNA isolation: 1 M Tris–Cl, 0.5 M EDTA, 5 M NaCl, 10% SDS.

2.3 Nucleofection of Vectors into hiPSCs in a Feeder-Free Condition

Human recombinant Laminin-521 (BioLamina). DPBS, calcium, magnesium (Gibco). StemFlex (Gibco): Mix StemFlex Basal Medium with StemFlex supplement, and divide into aliquots. Store at 20
C. Corning 100 mm TC-Treated Culture Dishes (Corning).

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Puromycin-Resistant Mouse Embryonic Fibroblasts, Day E13.5 (STEMCELL Technologies). DPBS, no calcium, no magnesium (Gibco). 0.4 mg/mL Mitomycin C (ACROS ORGANICS): Dissolve 2 mg in 5 mL of prewarmed DPBS, no calcium, no magnesium. 0.05% Trypsin-EDTA (Gibco). 10% FBS media. Accutase cell detachment solution (STEMCELL Technologies). Nucleofector 2b (Lonza). Human Stem Cell Nucleofector Kit 1 (Lonza). Rho-associated protein kinase (ROCK) inhibitor (Y-27632, Millipore). NHEJ inhibitor-SCR7 (Xcess Biosciences Inc.) [9]. Puromycin dihydrochloride (Life Technologies). 2.4 Neural Stem Cell Differentiation

Disposable Cell Lifter (Fisher Scientific). 20 μg/mL human recombinant basic fibroblast growth factor (bFGF): Dissolve 50 μg in 2.5 mL of 10 mM Tris base buffer, pH 7.6, and divide into aliquots. Store at 20
C. 100 μg/mL human recombinant epidermal growth factor (EGF): Dissolve 500 μg in 5 mL of 10 mM Tris base buffer, pH 7.6, and divide into aliquots. Store at 20
C. NSC media: Prepare 50 mL of NN1 media (NeoNeuron), and add 50 μL of 20 μg/mL bFGF (final 20 ng/mL) and 10 μL of 100 μg/mL EGF (final 20 ng/mL). Store at 4
C. Corning 60 mm TC-Treated Culture Dish (Corning). 0.05% Trypsin-EDTA (Gibco). Trypsin neutralizer solution (Gibco). Corning 24 well TC-Treated Multiple Well Plates (Corning). 10 poly-L-ornithine: Prepare 0.16 mg/mL of poly-L-ornithine as 10 times concentrated, and divide into aliquots. Store at 20
C. Differentiation of dopaminergic neurons: Prepare 25% of NN1 media (NeoNeuron) and 75% of DIF1 (NeoNeuron), 10 ng/ mL of bFGF, 200 mM ascorbic acid, and 1% of FBS. Store at 4
C (see Note 1).

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Methods

3.1 Construction of gRNA Expressing Vector and Donor Vector

1. Construction of gRNA Expressing Vector. (a) Search the candidate gRNAs using online gRNA searching website, such as CRISPRdirect [10] or refer to published paper (see Note 2). (b) Order gRNA oligonucleotides with additional sequences for the connection with BbsI cleaved ends of backbone plasmid. Forward: CACC (20 bp gRNA sequences) CT. Reverse: AAACAG (20 bp reversed gRNA sequences) (c) Annealing the gRNA Oligonucleotides Using PCR. Mix 24.5 μL of each oligonucleotide (100 μM) with 1 μL of 5 M NaCl, and total volume is 50 μL. Set the mixture on the PCR machine and annealing by operation as 99
C 5 min, 95
C 5 min and 5
C every 5 min to 20
C, and hold at 4
C. Dilute the annealed oligonucleotides 1000 times with TE buffer. (d) Ligation to pX458 Plasmid. Linearize pX458 by BbsI and purify by electrophoresis and gel extraction. Mix 2 μL of the annealed oligonucleotides, appropriate amount of linearized pX458, 10XT4 ligase buffer, and T4 DNA ligase, and react on an ambient temperature for 1 h. Apply into a competent cell for the transfection, and confirm the insertion of gRNA sequences by sequencing. 2. Construction of Recombination.

Donor

Vector

for

Homologous

(a) Linearize AAVS1-hPGK-PuroR-pA donor plasmid with Pme I and EcoR I, and extract backbone plasmid by electrophoresis-gel extraction. (b) Prepare wild-type genomic DNA (see Note 3) and pX458 plasmid as PCR template to clone homologous arms and T2A-GFP-pA. Set the PCR primers having additional 16 bp homologous sequences with the linearized AAVS1 hPGK-PuroR-pA donor plasmid at the 50 end (Fig. 1, dotted  line above a forward and c reverse primer). l

If homologous arms include the gRNA target sequences, divide into two PCR products, and set PCR primers inserting one nucleotide mutated on PAM sequences (NGG to NXG or NGX) (see Note 4). Perform PCR using a and b primer set with genomic DNA and c primer set with pX458 plasmid, and extract PCR products by electrophoresis-gel extraction (see Notes 5 and 6). Using extracted PCR products (a and b products) as template, perform the next PCR

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123

Fig. 1 Donor vector illustrates where it targets on TH genomic locus. T2A-GFP-pA and hPGK-PuroR-pA are integrated in place of stop codon. Blue line is gRNA target sequence, red is PAM sequence, and black head on the TH-Left arm is the mutation on PAM sequence. a, b and c primer sets are for the PCR to clone TH-Left arm-T2A-GFP-pA. a forward primer and c reverse primer have 16 bp sequence homologous with backbone plasmid

(a forward + b reverse primer set) having mutation on PAM sequences in the TH-Left arm. Using PCR product (TH-Left arm and c product) as template, perform PCR (a forward + c reverse primer set), and extract TH-Left arm-T2A-GFP-pA product (Fig. 1). (c) Calculate mole number of inserting PCR product and linearized backbone plasmid. Mix the same mole amount of insert and plasmid. Add Gibson assembly reagent at the same volume of total DNA mixture, and react on 50
C for 15 min. Add ddH2O 4 volumes of total DNA-Gibson mixture, and apply 1/10 volume to transfection into the competent cell. Confirm the insertion by PCR and sequencing. (d) Insert hPGK-PuroR-pA and TH-Right arm as the same process with TH-Left arm-T2A-GFP-pA. After adding TH-Left arm-T2A-GFP-pA, Not I was used for linearization, and TH-Right arm was inserted (see Note 7). Xba I-recognition sequences were included on the forward primer of TH-Right arm to linearize TH-Left arm-T2AGFP-pA-TH-Right arm donor template. After Xba I cut of TH-Left arm-T2A-GFP-pA-TH-Right arm donor template, hPGK-PuroR-pA was inserted (see Note 7). Confirm the insertions by PCR and sequencing. 3.2 Verification of Optimal gRNA

1. Purify the constructed pX458gRNA vectors using Miniprep kits according to the manufacturer’s instructions. 2. 1 day before transfection, culture the HEK293FT cells (see Note 8) in 10% FBS media. Plate HEK293FT cells as 1  106 on 60 mm dishes as the number of validating pX458gRNAs.

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Fig. 2 Illustration of T7E1 assay and example of gel electrophoresis. DSB occurs in the targeted genomic region and is repaired by NHEJ, leading Indel sequences. Reannealing of PCR product from Indel sequences and wild sequences generates homoduplex strands and heteroduplex strands. T7E1 cleaves the mismatched strands. Lane 1 is the result of gRNA#01, and lane 2 is the result of control. Upper band near 750 bp indicates uncleaved PCR products containing homoduplex of Indel and wild strands. Lower two bands indicate cleaved PCR products

3. Prepare 150 μL of Opti-MEM and add 11.25 μL of Lipofectamine 2000, and separately prepare 150 μL of Opti-MEM and add 3.75 μg of pX458gRNA. Let stand for 5 min. Then mix Lipofectamine mixture with DNA mixture, and let stand for 20 min. Add Lipofectamine-DNA mixture into the HEK293FT cells plated the previous day. Mix by moving back and forth gently. The next day, change the media. 4. Two days after the transfection, check whether the GFP is expressing (see Note 9). Trypsinize, neutralize, and pipette to make single cells for FACS sorting. Collect more than 2  105 GFP-positive cells and lysed with genomic DNA lysis buffer. Isolate genomic DNA by phenol-chloroform extraction (see Note 3). 5. Perform PCR using the genomic DNA from GFP-sorted cells for 40 cycles to expand the region including gRNA-targeted genomic locus. Simultaneously perform the same PCR using wild HEK293FT gDNA for control. 6. Reanneal the PCR products in a condition of 95
C 5 min, 95–85
C 2
C/s, and 85–25
C 0.1
C/s and hold at 4
C. 7. Add appropriate amount of 10XNEB2 buffer and 0.5 μL of T7E1 into the reannealed PCR product. Incubate at 37
C for 2 h. 8. Check cleaved band by gel electrophoresis (Fig. 2).

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1. Linearize donor vector using Nco I (see Note 7), and purify linearized donor vector and selected pX458gRNA vector. 2. Prepare confluent iPSCs cultured on StemFlex feeder-free condition. Add 2 mL of DPBS containing calcium and magnesium with 0.5 μg/cm2 of Laminin-521 into 60 mm dish. Incubate at 4
C overnight. 3. Aspirate the DPBS-Laminin-521 from the 60 mm dish. Add fresh StemFlex media containing 10 nM of ROCK inhibitor into Laminin-521-coated 60 mm dish, and stand by at the incubator. 4. Make iPSCs into single cells using Accutase cell detachment solution and neutralize. Count the cell number. 5. Mix 82 μL of Stem Cell Nucleofector solution with 18 μL of Nucleofector supplement. Add 5 μg of pX458THgRNA vector and 5 μg of donor vector for 2  106 cells. 6. Spin down 2  106 cells and remove the supernatant. Remove the supernatant completely using P200 pipette. 7. Add DNA mixture into the cell pellet and suspend. Transfer DNA-cell mixture into a cuvette, and apply program A-023 in Nucleofector 2b. 8. Immediately transfer the nucleofected cells to the prewarmed Laminin-521-coated medium containing 60 mm dish [12], and incubate for 2 days (see Note 10). 9. One day after the nucleofection, add 2 mL of DPBS containing calcium and magnesium with 0.5 μg/cm2 of Laminin-521 into 60 mm dish. Incubate at 4
C overnight. 10. Two days after the nucleofection, aspirate the DPBS-Laminin521 from the 60 mm dish. Add fresh StemFlex media containing 10 nM of ROCK inhibitor into Laminin-521-coated 60 mm dish. 11. Make the nucleofected cells into single cells using Accutase cell detachment solution. Neutralize. Sort GFP-positive cells by FACS in a sanitized condition at the level of culturing. 12. Plate the GFP-positive cells on the Laminin-521-coated 60 mm dish. Incubate for 2 days for the recovery from FACS sorting. 13. Change the media every 2 days with fresh media containing 0.5 μg/mL puromycin to select homologously recombinated clones. If the cell’s morphology does not look healthy after the FACS sorting, start puromycin selection several days later. Continue puromycin selection until the cells start to form colonies. 14. Select the colonies when they are enlarged enough to be selected.

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3.4 Confirmation of Cloned iPSCs

After selecting appropriately enlarged colony, expand and split cells for freezing and genomic DNA isolation to verify an integration of the donor vector, i.e., either precisely integrated or randomly integrated. Use either Southern blot or genome walking PCR kit to distinguish the integrations (see Note 11).

3.5 Neural Differentiation from Cloned iPSCs Containing TH Reporter [5]

1. Prepare a confluent iPSCs on 60 mm dish. At day 1, collect iPSCs by cell lifter, and spin down 200  g for 5 min.

3.5.1 Differentiation of iPSCs Containing TH-GFP Reporter to Neural Stem Cells

2. Remove the supernatant and add 1 mL of NSC medium. Suspend the pellet, and spin down 200  g for 5 min. 3. Remove the supernatant and add 10 mL of NSC medium. Resuspend and plate on T25 flask. Incubate for 3 days. At day 3 the cell clumps gradually make embryoid bodies. 4. Collect the floating cells and spin down 200  g for 5 min. Remove the supernatant, and add fresh NSC medium and plate on used T25 flask. Change the media every 2–3 days. 5. At day 17, collect the floating cells and clumps, and spin down 200  g for 5 min. Remove the supernatant and add 0.5 mL of Accutase cell detachment solution, and incubate for 3 min. Add 0.5 mL of trypsin neutralizer solution, and break down the cell clumps by pipetting gently using P1000 pipette. Add the used media and spin down 200  g for 5 min. 6. Remove the supernatant and add 10 mL of fresh NSC media. Suspend and plate on T25 flask. Repeat 17 day process until the embryoid bodies make the neurospheres and expansion.

3.5.2 Differentiation of Dopaminergic Neuron from NSCs

1. Prepare 24-well plate and add 0.45 mL of PBS with 0.05 mL of poly-L-ornithine (10). Incubate overnight at 37
C. 2. Collect the NSCs and spin down 200  g for 5 min. 3. Remove the supernatant for the next use, and add 1 mL of trypsin to the pellet. Incubate at 37
C for 5 min. 4. Neutralize with 1 mL of trypsin neutralizer, and break down the neurospheres by pipetting for 40 times. Add the media, and spin down 200  g for 5 min. 5. Remove poly-L-ornithine coating and wash with PBS twice. Proceed to the next step. 6. Remove the supernatant after the centrifuge. Add fresh conditioned media, and plate on the poly-L-ornithine-coated plate. 7. Change the media every day. The neurospheres will attach on the bottom in an hour. TH-positive cells will gradually appear in 3 days (see Note 12).

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4

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Notes 1. All reagents should be in an ambient temperature when use. 2. The cleaved nucleotide is the third nucleotide upstream from PAM sequence. Find gRNA target sequence as close to the stop codon as possible (TAG/CTA). 3. When isolating genomic DNA, it is preferable to remove RNAs and proteins by treatment of RNase and proteinase K before phenol-chloroform extraction. 4. To prevent donor template to be targeted by CRISPR-gRNA, it is preferable to add mutation on PAM sequence, called blocking mutation. When adding the mutation on PAM sequences, if the PAM sequences are on the coding region, consider not changing the amino acid. If the target sequence is not on the coding region, it is not necessary to consider [11]. 5. Be sure to linearize plasmid by electrophoresis after gel extraction as circular plasmid is not contaminated. 6. When constructing donor vector, complete the electrophoresis-gel extraction after every PCR so as not to contaminate whole genome as PCR template. 7. Be sure that restriction enzyme recognition sequences are not included in the region previously inserted to the donor vector in every step. 8. It is not necessary to use HEK293 cell for optimization of gRNA. iPSCs can be used simultaneously to confirm cleavage and modify gene as dividing FACS-sorted cells. 9. GFP will be expressed when pX458gRNA vector is transfected because GFP is connected with SpCas9 through T2A and will gradually disappear with cell division. 10. Sorting of GFP-positive cells 48 h after the nucleofection may support decreased frequency of randomly integrated clones. It is possible to skip FACS sorting and direct to puromycin selection, and pick a number of colonies. 11. Set the primers: (1) on the region outside of homologous arm and (2) in the homologous arm. 12. TH-positive cells can be observed by immunocytochemistry and confocal imaging.

Acknowledgment The authors thank members of the Daadi laboratory for the helpful support and suggestions. This work was supported by the Worth Family Fund, the Perry & Ruby Stevens Charitable Foundation and

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the Robert J., Jr. and Helen C. Kleberg Foundation, the NIH primate center base grant (Office of Research Infrastructure Programs/OD P51 OD011133), and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1 TR001120. Disclosures: Dr. Marcel M. Daadi is founder of the biotech company NeoNeuron. References 1. Daadi MM, Grueter BA, Malenka RC, Redmond DE Jr, Steinberg GK (2012) Dopaminergic neurons from midbrain-specified human embryonic stem cell-derived neural stem cells engrafted in a monkey model of Parkinson’s disease. PLoS One 7:1–11 2. Cui J, Rothstein M, Bennett T, Zhang P, Xia N, Pera RAR (2016) Quantification of dopaminergic neuron differentiation and neurotoxicity via a genetic reporter. Sci Rep 6:1–8 3. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2006) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872 4. Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, Hong H, Nakagawa M, Tanabe K, Tezuka K, Shibata T, Kunisada T, Takahashi M, Takahashi J, Saji H, Yamanaka S (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8:409–412 5. Liu G, Qu J, Suzuki K, Nivet E, Li M, Montserrat N, Yi F, Xu X, Ruiz S, Zhang W, Wagner U, Kim A, Ren B, Li Y, Goebl A, Kim J, Soligalla RD, Dubova I, Thompson J, Ill JY, Esteba CR, Sancho-Martinez I, Belmonte JCI (2012) Progressive degeneration of human neural stem cells caused by pathogenic LRRK2. Nature 491:603–607 6. Reinhardt P, Schmid B, Burbulla LF, Scho¨ndorf DC, Wagner L, Glatza M, Ho¨ing S, Hargus G, Heck SA, Dhingra A, Wu G, Mu¨ller S, Brockmann K, Kluba T, Maisel M, Kru¨ger R, Berg D, Tsytsyura Y, Thiel CS, Psathaki O, Klingauf J, Kuhlmann T, Klewin M, Mu¨ller H, Gasser T, Scho¨ler HR, Sterneckert J (2013) Genetic correction of a

LRRK2 mutation in human iPSCs links Parkinsonian neurodegeneration to ERK-dependent changes in gene expression. Cell Stem Cell 12:354–367 7. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308 8. Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller FC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851–857 9. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL (2015) Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol 33:538–542 10. Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas9 guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123 11. Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, Olsen KM, Gregg A, Noggle S, Tessier-Lavigne M (2013) Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533:125–129 12. Rodin S, Antonsson L, Hovatta O, Tryggvason K (2014) Monolayer culturing and cloning of human pluripotent stem cells on laminin-521based matrices under xeno-free and chemically defined conditions. Nat Protoc 9:2354–2368

Chapter 10 Single-Cell Library Preparation of iPSC-Derived Neural Stem Cells Jeffrey Kim and Marcel M. Daadi Abstract Single-cell RNA-seq technology allows for the identification of heterogeneous cell populations, measures stochastic gene expressions, and identifies highly variable genes. Thus, with this technology it is possible to identify relevant pathways involved in development or in disease progression. Herein, we describe a protocol to capture and process single-cell transcriptomes that will be used for RNA sequencing. This chapter discusses the use of the Fluidigm C1 System and Integrated Fluidic Circuit microfluidics system, TapeStation 4200, SMART-Seq v4, Nextera XT Library Preparation Kit, and AMPure XP beads. Key words Single cell, Neural stem cells, Fluidigm, C1, Library preparation, RNA-seq, Single-cell RNA-seq, Nextera XT, Next-generation sequencing, SMART-Seq

1

Introduction Next-generation sequencing (NGS) is a high-throughput and costeffective means of sequencing genomic material [1]. With NGS technology, sequencing whole transcriptomes has become possible. RNA sequencing (RNA-seq) is used to measure average expression levels for genes across a population of cells. It is useful for comparative transcriptomics and quantifying expression patterns. However, each cell is unique with its own gene expression profile. As bulk RNA-seq represents a large population of cells, there may be bias toward a subpopulation with high expression and therefore be insufficient when dealing with heterogeneous systems [2]. Averaging of these expressions does not represent the individual cells, and rare cell types may remain hidden. Single-cell RNA-seq (scRNAseq) is a relatively new technology that solves these issues with bulk RNA-seq. Unlike bulk RNA-seq, scRNA-seq measures the distribution of gene expression levels in single cells, which allows for the observation of cell-specific transcriptomic changes. scRNA-seq allows us to identify cell types in heterogeneous cell populations, measure the stochastic expression profile of individual cells, and

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_10, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Fig. 1 Workflow for single-cell RNA-seq using iPSC-derived NSCs with the C1 auto prep system. NSCs derived from iPSCs grow as neurospheres. Neurospheres are dissociated into single cells and loaded into the C1 IFC. Following the single-cell cDNA generation, libraries are amplified and indexed with the Nextera XT Library Preparation Kit. Generated libraries are then cleaned with AMPure XP beads and quality checked with the Agilent TapeStation 4200. Cleaned libraries are then sequenced with an Illumina-based next-generation sequencing platform. Raw reads are then demultiplexed and analyzed

identify highly variable genes across the sample population [3]. Furthermore, scRNA-seq enables the identification of pathways involved in cell development, physiology, or disease pathologies. Single-cell technology has found a niche in stem cell biology and can be used for studying cellular diversity, identifying subpopulations in heterogeneous culture systems and stem cell-based disease models. There are many methods available for capturing single cells for analysis. Manual cell picking, fluorescence-activated cell sorting (FACS) [4], magnetic-activated cell sorting (MACS) [5], laser capture microdissection (LCM) [6], or microfluidics [7] are some examples. Single-cell analyses tend to be challenging to properly perform in capturing single cells and in working with small-scale template material amplification for high-throughput sequencing. Importantly, having a reliable pipeline will ensure the single-cell analysis is reliable. To begin preparing the samples for sequencing, first extract genomic material (exome, transcriptome, or whole-genome) from the individual cells for the generation of cDNA libraries. This chapter covers the use of Fluidigm’s C1 Integrated Fluidic Circuits (IFC) microfluidic system with SMART-Seq v4 chemistry and Illumina Nextera XT Library Preparation Kit for the generation of sequencing libraries. It will cover every step from cell preparation to sequencing (Fig. 1).

2

Materials

2.1 Thawing Reagents

1. Warm Cell Wash Buffer, C1 Preloading Reagents, C1 Blocking Reagents, and C1 Harvest Reagent to room temperature. 2. Thaw C1 Suspension Buffer on ice and vortex well before use. 3. Thaw 5
Ultra Low First-Strand Buffer at room temperature. 4. Thaw all remaining reagents for first-strand cDNA syntheses (except enzyme) on ice. 5. Gently vortex each reagent and spin down. 6. Store all but 5
Ultra Low First-Strand Buffer on ice.

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Table 1 Serial dilution for cell buoyancy test

Cells (μL) Suspension reagent (μL)

50%

55%

60%

65%

70%

75%

80%

85%

20

10

11

12

13

14

15

16

17

0

10

9

8

7

6

5

4

3

Table 2 Dilution of spike-in mix Dilution

Spike-in Mix

Nuclease-free Water

1:10

1 μL undiluted

9 μL

1:100

1 μL of 1:10

9 μL

1:1000

1 μL of 1:100

9 μL

1:10000

1 μL of 1:1000

9 μL

1:100000

1 μL of 1:10000

9 μL

2.2 Cell Buoyancy Test

When utilizing the C1 microfluidic system, it is imperative that cells float in suspension and it is essential to test buoyancy of single cells. 1. Dissociate the stem cell colonies or clusters into single cells with enzyme of choice. 2. Centrifuge at 1000 rpm (200
g) for 5 min. 3. Perform a serial dilution of the cell suspension with Fluidigm’s Suspension Reagent. Refer to Table 1. 4. In a clear, flat-bottomed 384-well plate, add in 20 μL per well of the cell suspension mix. 5. Let suspensions sit for 10 min, and exam each well for even cell distribution throughout the entire volume of the well. 6. The best ratio of cell mix and suspension reagent will be used for the actual experiment.

2.3 Prepare RNA Spike-in Mix

2.4 Prepare 10
Reaction Buffer

The concentration of RNA spike-in varies depending on the cell type. To determine required dilution, consult pertinent literature. For neural stem cells, the concentration used is anywhere between 1:100000 and 1:400000. When using the ERCC RNA spike-in mix (ThermoFisher), first dilute the stock spike-in mix. Follow the series dilution in the table below (Table 2). Note that only 1 μL of spike-in is needed for the cell lysis mix. 1. Mix the following reagents and keep on ice. Refer to Table 3. 2. Pipet up and down to mix and spin down briefly. Keep on ice until ready to use.

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Table 3 10
Reaction Buffer mix Component

Volume (μL)

10
Lysis Buffer

19

RNAse Inhibitor

1

Total

20

Table 4 Cell lysis mix Component C1 Loading Reagent (20
) 0

3 SMART-Seq CDS Primer II A (12 μM) Nuclease-free Water 10
Reaction Buffer Total

Volume (μL) 1 2.4 14 2.6 20

Table 5 Reverse transcriptase mix Component C1 Loading Reagent (20
) 5
Ultra Low First-strand Buffer (RNase-free)

1.2 11.2

SMART-seq v4 Oligonucleotide (48 uM)

2.8

Rnase Inhibitor

1.4

Nuclease-free Water

9.8

SMARTScribe Reverse Transcriptase (100 U/μL)

5.6

Total

2.5 Prepare Cell Lysis Mix

Volume (μL)

32

1. Mix the following in a tube labeled “lysis” and keep on ice (see Note 1). Refer to Table 4. 2. Pipet the cell lysis mix up and down a few times to mix and spin down. Keep on ice until ready to use.

2.6

Prepare RT Mix

1. Mix together all except SMARTScribe Reverse Transcriptase in a tube labeled “RT,” and keep on ice. Add in SMARTScribe Reverse Transcriptase just before pipetting RT mixture into IFC. Refer to Table 5. 2. Gently vortex and spin down. Keep on ice until use (see Note 2).

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Table 6 PCR mix Component

Volume (μL)

C1 Loading Reagent (20
)

4.5

Nuclease-free Water

4.4

PCR Primer II A (12 μM)

3

SeqAmp PCR Buffer (2
)

75.2

SeqAMP DNA Polumerase (1.25 U/μL) Total

2.9 90

Fig. 2 Integrated Fluidic Circuits (IFC) priming reagent map 2.7

Prepare PCR Mix

1. Mix together in a tube labeled “PCR” and keep on ice. Refer to Table 6. 2. Gently vortex for 3 s and centrifuge briefly. Keep on ice until ready to use.

3 3.1

Methods Priming IFC Chip

Required reagents: C1 Harvest Reagent, C1 Preloading Reagent, C1 Blocking Reagent, and Cell Wash Buffer. Avoid creating air bubbles in inlets of chip during the priming process. 1. Place C1 IFC onto the provided IFC map. Refer to Fig. 2 for Subheading 3.1.

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2. Add 200 μL of C1 Harvest Reagent into the accumulators which are marked with red circles (2 total). Push down on the O-ring with the pipet tip and inject reagent. 3. Swirl IFC gently to distribute the reagent evenly. 4. Pipet 20 μL of C1 Harvest Reagent into inlets marked with solid red circles on each side of the accumulators (36 total). 5. Pipet 20 μL of C1 Harvest Reagent into the two inlets marked with small red circles in the middle of the outside columns of inlets on each side of the IFC (4 total). 6. Pipet 15 μL of C1 Blocking Reagent into the cell inlet and outlet marked with yellow dots (2 total). 7. Peel off white tape on bottom of IFC. Do not peel the four pieces of tape that cover the harvest inlets. 8. Place the IFC into the C1 system. 9. Run the SMART-seq v4: Prime (1861
/1862
/1863
) script. 10. Priming takes approximately 10 min. 11. When the Prime script has finished, tap EJECT to remove the primed IFC from the instrument (see Note 3). 3.2

Cell Preparation

This process can be performed while the IFC chip is being primed. 1. Isolate neurospheres from culture. 2. Centrifuge to form a pellet and remove supernatant. 3. Wash with phosphate-buffered saline (PBS). 4. Centrifuge to form a pellet and remove supernatant. 5. Add 1 mL of Accutase and resuspend the pellet. 6. Incubate the suspension at 37  C for 5 min. 7. Triturate cell suspension and add 5 mL of fresh media. 8. Centrifuge to form pellet and remove supernatant. 9. Resuspend cells with 1 mL of fresh media. 10. Count the cells and measure viability with method of choice e.g. Countess (ThermoFisher). 11. Use 10 μL of the cell suspension with 10 μL of Trypan blue. Load the suspension into cell counting chamber slides and quantify with Countess. 12. Prepare a cell suspension in native 66,000–333,000 cells/mL (see Note 4).

medium

of

13. Suspend the cells in a final volume of 0.5–1 mL to ensure enough cells are viable for the IFC and tube controls. 14. Prepare the cell mix by combining cells with suspension reagent at the ratio optimized in advance (see Note 5).

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Fig. 3 IFC loading cell map 3.3

Loading Cells

1. Use pipet to remove blocking reagent from inlets (marked with teal and gray dots). Refer to Fig. 3 for Subheading 3.3. 2. Set a pipet to 12 μL and slowly pipet cell mix 5–10 times to mix and dissociate any clumps (see Note 6). 3. Pipet 24 μL of C1 Preloading Reagent into Inlet 2 on the left side of chip (marked with purple dot). 4. Pipet 7 μL of Cell Wash Buffer into Inlet 6 on the right side of the chip (marked with pink dot). 5. Pipet 5 μL of cell mix into inlet marked with teal dot (see Note 7). 6. Place the IFC into the C1. 7. Run the SMART-Seq v4: Cell Load (1861
/1862
/1863
). 8. When the script has finished, tap EJECT to remove the IFC from the C1 system. 9. At this point, check each capture chamber under a phase contrast microscope, and note which chambers exhibit successful single-cell capture and which were unsuccessful.

3.4 Run Lysis, Reverse Transcription, and PCR on the C1

1. Aspirate solution in the flowthrough outlet (marked with gray dot). Refer to Fig. 4 for Subheading 3.4. 2. Pipet 180 μL of C1 Harvest Reagent into the four reservoirs marked with large solid red rectangles. 3. Pipet 7 μL of lysis Mix A in Inlet 3 (marked with an orange dot). 4. Pipet 8 μL of RT Mix in Inlet 4 (marked with a yellow dot). 5. Pipet 24 μL of PCR Mix C in Inlets 7 and 8 (marked with blue dots). 6. Place the IFC into the C1 system. 7. Run the SMART-Seq v4: Sample Prep (1861
/1862
/ 1863
) script. Run time for this operation is 8 h and can be performed overnight (see Note 8).

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Fig. 4 IFC sample prep map 3.5 Harvest Amplified Products

Pre-warm C1 DNA Dilution Reagent to room temperature. If running the Sample Prep script overnight, a 96-well plate with 10 μL aliquots of dilution reagent can be prepared the day before. This should be sealed and put into 4  C overnight and pre-warmed to room temperature before using. 1. When the SMART-Seq v4: Sample Prep script has finished, tap EJECT to remove the IFC from the instrument. 2. Transfer the C1 IFC to a post-PCR lab environment. 3. Label a new 96-well plate as “Diluted Harvest Plate.” 4. Aliquot 10 μL of C1 DNA Dilution Reagent into each well of the diluted harvest plate. 5. Carefully pull back the tape covering the harvesting inlets of the IFC using the plastic removal tool. 6. Using an eight-channel pipet (set to 3.5 μL), pipet the harvested amplicons from the inlets (see Note 9). Use the bottom schematic to map the harvest (Fig. 5). Start with the top left well and work to the right. Then move to the well below the starting point and again work to the right. This should complete the harvest if using an eight-channel pipet. 7. Pipet the entire volume of C1 harvest amplicons into the 10 μL of C1 DNA Dilution Reagent in each well of the “Diluted Harvest” plate. 8. Seal and vortex the plate for 20 s, and then centrifuge at 350
g for 1 min. 9. Samples can be stored up to a week in 4  C or long term in 20  C.

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Fig. 5 Harvesting sequence map Table 7 cDNA sample dilution for sequencing cDNA Sample dilution

1:2

1:3

1:4

1:5

1:6

1:8

1:10

1:12

Volume of C1 harvest reagent required

2 μL

4 μL

6 μL

8 μL

10 μL

14 μL

18 μL

22 μL

3.6 cDNA Sample Dilution

Measure the concentrations of the libraries using a NanoDrop or similar device, such as PicoGreen assay. If the NanoDrop is not sensitive enough to detect low concentrations, then dilute harvested amplicons to optimal working concentration. The optimal concentration for Nextera XT library preparation is 0.10–0.30 ng/μL. 1. Label a new 96-well PCR plate as “Diluted for Sequencing.” 2. Pipet the appropriate amount of C1 Harvest Reagent to each well of the plate listed in the table per determined sample dilution (Table 7). 3. Transfer 2 μL of the harvest sample from the “Diluted Harvest” plate to the “Diluted for Sequencing” plate. 4. Seal the plate with adhesive film. 5. Vortex at medium speed for 20 s, and centrifuge at 1500 rpm (450
g) for 1 min.

3.7 Tagment Genomic DNA

Use the following tagmentation program for the thermal cycler: Choose the preheat lid option. 55  C for 5 min. Hold at 10  C

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1. Thaw Amplicon Tagment Mix on ice. 2. Warm Tagment DNA Buffer and NT Buffer to room temperature. Vortex NT Buffer if precipitate is present. 3. Mix reagents by inverting thawed tubes 3–5 times followed by brief centrifugation. 4. Label a new 96-well PCR plate as “Library Prep.” 5. In a 1.5 mL PCR tube, combine the components of the tagmentation premix. Each sample will require 2.5 μL of Tagment DNA Buffer and 1.25 μL Amplification Tagment Mix. Make a master mix with the calculated amount of reagents for the number of samples. 6. Vortex gently for 20 s, followed by brief centrifugation. 7. Aliquot equal amounts of premix into each tube of an eighttube strip. 8. Using an eight-channel pipet, pipet 3.75 μL of the premix into each well of the “Library Prep” PCR plate. 9. Transfer 1.25 μL of each diluted sample from the “Diluted for Sequencing” plate to the “Library Prep” plate. 10. Seal and vortex the plate at medium speed for 20 s. Then centrifuge at 2000 rpm (800
g) for 5 min to remove any air bubbles. 11. Place the plate onto the thermal cycler and run the tagmentation program. Immediately proceed to the next step once the thermal cycler reaches 10  C. 12. Aliquot equal amounts of NT Buffer into each tube of an eighttube strip. 1.25 μL of NT Buffer will be required for each sample. 13. Once the thermal cycler again reaches 10  C, pipet 1.25 μL of NT Buffer into each of the samples for neutralization. 14. Seal and vortex the plate at medium speed for 20 s, and centrifuge at 2000 rpm (800
g) for 5 min. 3.8

Amplify Libraries

Use the following amplifying program for the thermal cycler. Choose the preheat lid option: 72  C for 3 min 95  C for 30 s 12 cycles of: 95  C for 10 s 55  C for 30 s 72  C for 30 s. 72  C for 5 min Hold at 10  C.

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1. Thaw Index 1 (N7) and 2 (S5) primers at room temp for 20 min. Invert and centrifuge briefly. 2. Thaw Nextera PCR Master Mix on ice for 20 min. 3. Aliquot equal volumes of Nextera PCR Master Mix into each tube of an eight-tube strip. Each sample will require 3.75 μL of master mix so calculate accordingly. 4. Using an eight-channel pipet, pipet 3.75 μL of master mix into each well of the “Library Prep” plate. 5. Arrange Index 1 (N7) adapters in columns 1–12. 6. Arrange Index 2 (S5) adapters in rows A–H. 7. Add 1.25 μL of each Index 1 (N7) adapter down each column. Replace the cap on each i7 adapter tube with a new orange cap (see Note 10). 8. Add 1.25 μL of each Index 2 (S5) adapter across each row. Replace the cap on each i5 adapter tube with a new white cap. 9. Seal and vortex plate at medium speed for 20 s. Then centrifuge at 2000 rpm (800
g) for 2 min. 10. Place on the thermal cycler and run the amplifying program (see Note 11). 11. Amplified products can be store long term in 20  C. 3.9 Clean Up Libraries

To perform library cleanup with AMPure XP beads, a magnetic separation device for 1.5 mL tubes will be needed. Before each use, bring bead aliquots to room temperature for at least 30 min. Make sure to mix well. 1. Prepare fresh 70% EtOH (see Note 12). About 800 μL per tube will be needed. 2. Aliquot AMPure XP beads into 1.5 mL tubes, and bring to room temperature and vortex for 1 min. 3. Pool the libraries by pipetting the volumes designated in the table below into a 1.5 mL tube (Table 8). 4. Add the designated amount of AMPure XP beads to the pooled libraries. 5. Pipet mixture slowly five times to mix. 6. Incubate bead mixture at room temperature for 5 min. 7. Briefly spin down the mixture, and collect the mixture from the side of the tube. 8. Place the tube on a magnetic stand for 2 min, until the liquid appears completely clear and no beads are present in the supernatant. 9. While the tube is left on the magnetic stand, pipet out the supernatant and discard.

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Table 8 Library cleanup with AMPure beads Number of libraries pooled

Volume per library (μL)

Total pool volume (μL)

AMPure bead volume (μL)

8

4

32

29

12

4

48

44

16

2

32

29

24

2

48

44

32

1

32

29

48

1

48

44

96

1

96

87

10. Keep the tube on the magnetic stand, and add 180 μL of 70% EtOH to the sample without disturbing the beads. 11. Wait for 30 s and carefully pipet out the supernatant and discard. 12. Repeat the EtOH wash one more time, but this time do not remove supernatant. 13. Remove the tube from the magnetic stand and spin down briefly. 14. Place the tube back on the magnetic stand for 30 s, and then remove all remaining ethanol. 15. Leave the tube on the magnetic stand, and air-dry the beads for 10–15 min or until the pellet is no longer shiny but before a crack appears (see Note 13). 16. Once the pellet is properly dried, add the designated amount of C1 DNA Dilution Reagent according to the table below (Table 9). 17. Remove the tube from the magnetic stand and vortex for 3 s to mix. 18. Incubate the sample at room temperature for 2 min to rehydrate the pellet. 19. Spin down the sample briefly, and place back on the magnetic stand until the solution becomes clear. 20. Transfer the supernatant to another tube. This will contain the purified cDNA libraries. 21. Repeat steps 4–15. 22. Elute the pellet with the following volumes of C1 DNA Dilution Reagent. Refer to Table 10. 23. Remove the tube from the magnetic stand and vortex for 3 s.

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Table 9 First elution of pellet during library cleanup Number of libraries pooled

C1 Dilution Reagent volume (μL)

8

32

12

48

16

32

24

48

32

32

48

48

96

96

Table 10 Second elution of pellet for library cleanup Number of libraries pooled

C1 Dilution reagent volume (μL)

8

48

12

72

16

48

24

72

32

48

48

72

96

144

24. Incubate the sample at room temperature for 2 min. 25. Place the tube back onto the magnetic stand until the supernatant becomes clear. 26. Carefully transfer the supernatant to a new 1.5 mL tube labeled “cleaned library.” 3.10 Quality Check Libraries

1. Allow Genomic DNA Reagents to equilibrate at room temperature for 30 min. 2. Prepare tube strip or 96-well plate. Add 1 μL of Genomic DNA Ladder to 10 μL of Genomic DNA Sample Buffer into the A1 position of a tube strip or 96-well plate. Add 1 μL of cDNA to 10 μL of Genomic DNA Sample Buffer (see Note 14). 3. Vortex at 2000 rpm for 1 min and spin down samples. 4. Power on the laptop and wait for it to be fully booted, and launch the 4200 TapeStation Control Software.

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5. Turn on the TapeStation 4200 and allow the machine to initialize. 6. Open the lid and clean out any tip waste and used ScreenTape devices. 7. Install the tip rack and remove the lid. 8. Flick the ScreenTape device to move any air bubbles into the buffer chamber. 9. Place first ScreenTape into the ScreenTape nest. The label should be facing to the front. 10. Insert extra ScreenTape into racks (see Note 15). 11. Centrifuge samples to remove air bubbles. 12. Insert sample tube strips and remove the lids (see Note 16). 13. In the software, select samples for analysis. Ladder is provided in location A1 of the samples. 14. Start the run. 15. Results will appear when the run has finished. Results can be saved as a pdf. 16. Pooled libraries can now be sent for sequencing.

4

Notes 1. If using an RNA spike-in, add 1 μL of RNA spike mix instead of C1 loading reagent. 2. Do not vigorously vortex the RT mixture. 3. After priming the IFC, there is up to 1 h to load the IFC with the C1 system. 4. The recommended concentration range ensures that a total of 200–1000 cells are loaded into the IFC. 5. Many cell types use the standard suspension ratio of 3:2. 6. Do not vortex and avoid bubbles. 7. Pipet up to 20 μL of cell mix, but only 5 μL will enter the IFC. 8. Harvest should be performed as soon as the Sample Prep script has completed. There is a possibility of evaporation if left for more than 8 h. Designate the time to complete the operation when setting up the script. 9. It is imperative to use a well-calibrated eight-channel pipet to harvest as much of the amplified product as possible. Replace tips after each use. Also, it is absolutely important to remove the samples in order according to the map provided by Fluidigm. This can be found in their written protocol for the C1. This is to ensure that the harvested amplicons can be traced back to the sample IDs.

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10. It is important to replace the cap of the index tubes with a new cap after every use. 11. At this point, it is a safe stopping point. If stopping, seal the plate, and store at 2–8  C for up to 2 days. Alternatively, leave on the thermal cycler overnight. 12. It is important to make fresh 80% EtOH every time. 13. If the pellet is under-dry, there will still be ethanol present in the sample. This will reduce the recovery yield. Also, if the pellet is overdry, it will take longer than 2 min to rehydrate and may also reduce yield. Keep an eye on the pellet for a matte finish. 14. For 16 or more samples, use 2 μL of Genomic DNA Ladder and 20 μL of Genomic DNA Sample Buffer. If using a 96-well plate, apply a foil seal to the sample plate. 15. Only use ScreenTape devices of identical assays. 16. Be sure not to create bubbles when removing the lids. If using the foil-sealed 96-well plate, do not remove the foil.

Acknowledgment The authors thank members of the Daadi laboratory for the helpful support and suggestions. This work was supported by the Worth Family Fund, the Perry & Ruby Stevens Charitable Foundation and the Robert J., Jr. and Helen C. Kleberg Foundation, the NIH primate center base grant (Office of Research Infrastructure Programs/OD P51 OD011133), and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1 TR001120. Disclosures: Dr. Marcel M. Daadi is founder of the biotech company NeoNeuron. References 1. Behjati S, Tarpey PS (2013) What is next generation sequencing? Arch Dis Child Educ Pract Ed 98:236–238 2. Bengtsson M, Sta˚hlberg A, Rorsman P, Kubista M (2005) Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels. Genome Res 15:1388–1392 3. Islam S et al (2011) Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res 21:1160–1167

4. Gross A et al (2015) Technologies for single-cell isolation. Int J Mol Sci 16:16897–16919 5. Welzel G, Seitz D, Schuster S (2015) Magneticactivated cell sorting (MACS) can be used as a large-scale method for establishing zebrafish neuronal cell cultures. Sci Rep 5:7959 6. Datta S et al (2015) Laser capture microdissection: big data from small samples. Histol Histopathol 30:1255–1269 7. Bhagat AAS et al (2010) Microfluidics for cell separation. Med Biol Eng Comput 48:999–1014

Chapter 11 Bioinformatics Analysis of Single-Cell RNA-Seq Raw Data from iPSC-Derived Neural Stem Cells Jeffrey Kim and Marcel M. Daadi Abstract This chapter describes a pipeline for basic bioinformatics analysis of single-cell sequencing data (see Chap. 10: Single-Cell Library Preparation). Starting with raw sequencing data, we describe how to quality check samples, to create an index from a reference genome, to align the sequences to an index, and to quantify transcript abundances. The curated data sets will enable differential expression analysis, population analysis, and pathway analysis. Key words Single cell, Neural stem cells, Fluidigm, C1, Bioinformatics, Single-cell analysis, Singlecell RNA-seq, RNA-seq, Singular, SCDE, Kallisto, Sleuth, DAVID, BBDuk, FASTQC

1

Introduction The general rule of thumb for RNA-seq analysis is to first quality check the raw sequences and remove those of low quality. Then the sequences are aligned to an index built from a reference genome. After the quantification of transcript abundances, differential expression may be observed and expression pathways may be analyzed. RNA-seq analysis can be performed as genome guided or de novo. De novo analysis is performed without a reference genome due to unknown or incomplete references. This protocol utilizes a human reference genome and therefore will follow a genomeguided pipeline. Alignment of reads for RNA-seq covers noncontinuous portions of reference genomes that result from transcript splicing. Therefore, alignment tools are designed to find optimal alignment of noncontinuous sequences. Herein, we utilize a lightweight pipeline that can be used on a standard desktop computer. This pipeline is designed to be relatively simple for a beginner in the field of bioinformatics. Subsequent analysis described in this pipeline was written with Mac OS X and under the assumption that paired-end sequencing data is being used. Lines starting with “$” will be typed into the terminal or shell, while lines starting with “>”

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_11, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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will be used in R. When designating the location of a file, /path/ to/ notation will be used. Please note the provided text /path/to/ is not the actual path to be used which is entirely dependent on the physical location within a user’s hard drive.

2

Materials This section will cover the tools and their installation, necessary for single-cell analysis pipeline.

2.1 Computer Specifications

When performing bioinformatics analysis, a Unix-based operating system, such as Linux or Mac OS X, is recommended. Unix allows for the use of command-line-based tools that can be automated using custom scripts. Command-line tools can be accessed through a shell. The shell will be used to install and use command-line tools that perform index building, alignment, and quantification of raw sequencing data. The following protocols will be utilizing shell on Mac OS X. Also, consider the computer strength since index building and alignment require a lot of processing power. Certain processes can be sped up by partitioning more CPU cores to a certain task. Another limiting factor when analyzing is computer storage space. As sequencing data creates very large files, it is easy to run out of hard drive space. Space may be managed as needed depending on the amount of data generated. If using a Windows PC, there are ways to utilize command-line tools. However, for ease of access for beginners in bioinformatics, a Unix-based computer with adequate storage is preferable.

2.2

RStudio

RStudio is an open source software that uses the R framework. Its graphical interface allows for easy usage of R. However, some features in the R packages discussed in this protocol do not work with RStudio and therefore must be performed through normal R. To install RStudio, first install R, which can be found in the following website: https://cran.r-project.org/. Download R for the relevant platform. Once installed, proceed to download RStudio from the following website: https://www.rstudio.com/ products/rstudio/download/.

2.3

FASTQC

When first obtaining raw sequencing data, check the quality of the sequencing experiment. Installation of this program does not require the use of command line. This program requires Java 7 or higher to run. Website to download FASTQC: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.

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BBDuk

147

BBDuk is a trimming tool that is part of the BBTools toolset. BBTools has a variety of other bioinformatics tools; however, here only BBDuk is used. Download it from sourceforge.net/projects/ bbmap/. Move the downloaded file to a working directory of choice. To install, go to the terminal or shell and change to the working directory and then input the following command: $ tar --xvzf BBMap_(version).tar.gz

This will create a subfolder named bbmap, which contains the scripts and files needed to use BBDuk. 2.5

Kallisto

Kallisto is a program for quantifying transcript abundances through pseudoalignment for rapid determination of compatibility of reads. Kallisto is also used to build an index from a reference genome. To install kallisto, use the following commands in shell: $ ruby --e “$(curl --fsSL https://raw. githubusercontent.com/Homebrew/install/master/ install)” $ brew tap homebrew/science $ brew install kallisto

2.6

Sleuth

Sleuth is an R package designed for usage downstream of Kallisto. To install Sleuth use the following packages in R: > source(“http://bioconductor.org/biocLite.R”) > biocLite(“rhdf5”) > install.packages(“devtools”) > devtools::install_github(“pachterlab/sleuth”)

2.7

Singular

Singular is an analysis toolset offered gratis through Fluidigm. Its ease of use through a graphical interface makes it easy for beginners in bioinformatics to perform basic analysis. However, this R package does not work with Mac computers. Singular analysis toolset software and practice sets can be downloaded from www.fluidigm. com/software. To install the package, in R, go to the packages tab and click install packages from local files. Then, select the downloaded zipped file. Once installed, in R, type: > Library(fluidigmSC) > firstRun()

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SCDE

SCDE is an R package used in the statistical analysis of single-cell RNA-seq data. Use this to observe differential expression across samples. To install SCDE use the following packages in R: > source(“https://bioconductor.org/biocLite.R”) > biocLite(“scde”)

2.9

3

DAVID

To use DAVID, proceed to the following URL: david.ncifcrf.gov.

Methods

3.1 QC and Trimming of Raw Sequences

1. Load fastq files into FASTQC. 2. Observe per base sequence quality. If the curve dips below a Phred score of 28, the sequence will require trimming (see Note 1). If more than half of the sequence is below 28, file may not be adequate for downstream analysis and should be removed. Any adapter sequences present in the overrepresented sequences will also need to be removed. 3. Perform the following command in terminal or shell. For the inputs, direct the pathway to the location of files. For pairedend sequencing data, provide two inputs, one for each sample. The outputs will be designated with a new directory. The qtrim parameter designates the direction of the trim (either left or right). In the following example, right is selected with “r.” The trimq parameter designates the Phred score threshold where trimming should occur. Here we set the threshold to 28, which will trim the bases to the right of where the Phred score dips below 28. Perform the following command in terminal or shell to trim sequence: $ bbduk.sh in1¼/path/to/read1.fq in2¼/path/to/ read2.fq out1¼/newpath/to/clean1.fq out2¼/ newpath/to/clean2.fq qtrim¼r trimq¼28

4. Repeat this process with all pairs that fail the initial QC. 5. Repeat QC for trimmed sequences to verify the effectiveness of trimming. These sequences will be used downstream. 3.2

Index Building

1. Perform the following command in the terminal or shell. The generated index file will be output into the current directory. Change directory prior to this command. To generate an index: $ kallisto index --i nameOfChoice /path/to/cDNA. FASTA

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1. Perform the following command after building the index. Here we set the number of bootstraps to 100. This can be changed if desired. The input files are the trimmed fastq inputted as pairs. To perform pseudoalignment: $ kallisto quant --i /path/to/nameOfIndex -b 100 -o nameOfDirectoryOutput /path/to/input1_1.fastq / path/to/input1_2.fastq

2. To expedite the process of quantifying the samples, write a script to automate the process. Create a plain text file that lists the name of the fastq files. Then, create a plain text file with the following commands. Create a plain text file with the following: #!/bin/sh while read i;do echo$i kallisto quant -i /path/to/kallisto_index -b 100 -o / path/to/output_dir/${i} /path/to/files_dir/${i} 1_1.fastq /path/to/files_dir/${i}1_2.fastq done < filenames.txt

Name this file runkallisto.sh. To run the script, type the following in terminal or shell. To run the script, type into terminal or shell: $ . /runkallisto.sh

3. Alternatively, use the following command to perform the same function as “quant” but without bootstrap. First make a plain text file called “batch.txt,” which includes columns for #id, file1, and file2 names. Alternative method for pseudoalignment: $ kallisto pseudo -i kallisto_index -o output -b batch.txt

3.4 Filtering Transcript Abundances and Annotation with Sleuth

1. To begin, run the following commands to open necessary R packages. Load the following packages: > library(“sleuth”) > library(“biomaRt”)

2. The following commands will designate the pathway to the location of the kallisto files. Use .h5 files as they contain bootstraps. First designate the base directory.

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Jeffrey Kim and Marcel M. Daadi Set base directory: > base_dir sample_id kal_dirs s2c s2c s2c mart t2g t2g so so so so sleuth_live(so)

13. Go to the summaries tab and to kallisto table to view the transcript abundances. Save this table as it will be needed to generate a data matrix. Processed data can also be viewed for alignment QC metrics. 3.5 Analysis with Singular

1. Before analysis of filtered data can begin, transcript abundances must be compiled into a plain text matrix. To do this, simply use an excel spreadsheet and save as a txt file. The rows designate genes, while the columns designate sample names.

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2. Begin by attaching the package into R (see Note 2). Load fluidigmSC package: > library(fluidigmSC)

3. When performing the following command, a new command prompt will appear. Input the data matrix and sample sheet. The sample data sheet created in the previous section can be used. This command will detect outliers in the data set. Detect outliers: > identifyOutliers()

4. Use generated outliers.fso for analysis. Type the following: Perform auto analysis: > autoAnalysis()

Another command prompt will appear. This time, input outliers.fso and sample sheet. For genes of interest, choose defined in the expression file to designate all genes; otherwise, designate which genes or how many of the top variable genes will be represented. Then choose the destination for the output. autoAnalysis will display a PCA plot, tSNE plot, a hierarchal clustering heatmap, and violin plots. 5. Enter command to select custom colors and symbols: Select colors and symbols: > fldm_exp display3DPCAScore(pca ¼ fldm_pca, x_axis¼1, y_axis¼2, z_axis¼3, locate¼TRUE)

7. Enter command to create an “anova object” which will be used to generate a volcano plot. Create an anova object: > anova anova_gene_list volcano_gene_list saveData()

3.6 Analysis with SCDE: Differential Expression

Begin by attaching the necessary packages. Load the packages: >library(scde) >library(org.Hs.eg.db)

1. Assign a counts variable to the data matrix. Define counts variable: > counts sg names(sg) table(sg)

3. The following command is used to work around a possible error.

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Error fix: > counts 0 > table(valid.cells) > o.ifm <  o.ifm[valid.cells,]

6. The following command defines a grid of expression magnitude values on which the numerical calculations will be carried out. Here a grid of 400 points is used. Define grid of expression magnitude values: > o.prior groups names(groups) ediff head(ediff[order(ediff$Z, decreasing ¼ TRUE),])

10. Command to display top downregulated genes; “tail command”. Display top downregulated genes: > tail(ediff[order(ediff$Z, decreasing ¼ TRUE),])

11. Command to create a table with the results. Create a results table: > write.table(ediff[order(abs(ediff$Z), decreasing ¼ TRUE),], file ¼ “results.txt”, row. names ¼ TRUE, col.names ¼ TRUE, sep ¼ “\t”, quote ¼ FALSE)

12. Command provides a web browser application where differentially expressed genes can be browsed (see Note 3). Browse differentially expressed genes: > scde.browse.diffexp(ediff, o.ifm, counts, o. prior, groups ¼ groups, name ¼ “diffexp1”, port ¼ 1299)

13. Command to allow the observation of differential expression in a single gene of interest. View a single gene or interest: > scde.test.gene.expression.difference (“GENEname”, models ¼ o.ifm, counts ¼ counts, prior ¼ o.prior)

3.7 Analysis with SCDE: Pathway and Gene Set Overdispersion Analysis

1. Load the count matrix. Load counts matrix: > counts cd counts varinfo 0))

8. Translate gene names to ids. Translate gene names to ids: > ids rids app show.app(app, "TITLE",browse¼TRUE, port¼1468)

20. View the pathway according to gene ontology gene set of interest. View pathways of interest: > pagoda.show.pathways(c(“GO: NumbersOfInterest”), varinfo, go.env, cell. clustering ¼ hc, margins ¼ c(1,5), show.cell. dendrogram ¼ TRUE, showRowLabels ¼ TRUE, showPC ¼ TRUE)

3.8

DAVID Analysis

1. On the DAVID website, click “Functional Annotation” in the side bar. 2. Provide DAVID with a list of genes of interest. 3. Select an identifier of interest. In this case use ensembl_gene_id, ensembl_transcript_id, or official_gene_symbol. 4. Select the list type as gene list. 5. Submit list.

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6. Select Homo sapiens for species, or other relevant species. 7. Click annotation clustering to view enriched pathways. 8. To save the information, click download file. Then, copy and paste the information into an excel spreadsheet.

4

Notes 1. Phred quality score is the measurement of the quality of raw sequencing data. A Phred score dictates the probability of an incorrect base call. A score of 30 means that 1 in 1000 base calls is incorrect, while a score of 20 means 1 in 100 is incorrect. 2. Analysis with Singular will be done through normal R as certain functions may not properly work in RStudio. 3. The command does not work under RStudio and will crash the program. Instead use base R. 4. The command does not work in RStudio and must be performed with base R.

Acknowledgment The authors thank the members of the Daadi laboratory for their helpful support and suggestions. This work was supported by the Worth Family Fund, the Perry & Ruby Stevens Charitable Foundation and the Robert J., Jr. and Helen C. Kleberg Foundation, the NIH primate center base grant (Office of Research Infrastructure Programs/OD P51 OD011133), and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1 TR001120. Disclosures: Dr. Marcel M. Daadi is founder of the biotech company NeoNeuron.

Chapter 12 Assay for Assessing Mitochondrial Function in iPSC-Derived Neural Stem Cells and Dopaminergic Neurons Gourav Roy-Choudhury and Marcel M. Daadi Abstract Rapid and reliable assessment of mitochondrial bioenergetics is a vital tool in drug discovery studies aimed at reversing or improving mitochondrial dysfunction. Induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) carry and replicate the donor disease pathology and can be an ideal cellular model for phenotypic screening of compounds. Herein we describe the use of Seahorse XFe96 analyzer to assess mitochondrial functions in iPSC-derived NSCs for drug screening. Key words iPSCs, Mitochondrial functions, LRRK-2, Parkinson’s disease, Drug screening, Kinase inhibitors

1

Introduction Mitochondria play a critical role in cellular metabolism, reactive oxygen species (ROS) production, and apoptosis. Thus, the assessment of mitochondrial functions provides valuable insight into the physiology and bioenergetics of the cells. Mitochondrial dysfunction has been linked to the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and others [1]. Drugs targeting or improving mitochondrial functions could therefore be of interest for developing novel therapies. Investigation of these drugs requires rapid and reliable assessment of their effect on mitochondrial function. Current assays available for mitochondrial function can be broadly classified as either extracellular flux analyzers or complementary functional analyzers [2]. The extracellular flux analyzers enable a noninvasive quantification of mitochondrial respiration, whereas the complementary functional analyzers, such as ATP (adenosine triphosphate) assays, mitochondrial membrane potential assays, and ROS assay kits, measure specific functions of the organelle [3]. Although rapid, the complementary functional analysis methods are not extremely reliable due to the presence of artifacts

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_12, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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and low sensitivity. Furthermore, these methods are insufficient in delineating underlying mechanisms of mitochondria-associated metabolism. In contrast, extracellular flux analyzers provide realtime measurements of changes in mitochondrial respiration and bioenergetics. The development of 96-well plate systems, such as the Seahorse XFe96 analyzer, has made it possible to screen compounds for their effect on mitochondrial energetics in real time and short span [4–6]. The XFe96 analyzer has been widely used to measure the changes in mitochondrial respiration and glycolysis in different cell types such as: neurons [7, 8], astrocytes [9, 10], iPSCs [11–13], lymphocytes [14], and cancer cells [15]. We describe a detailed methodology of using induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) for drug screening with the XFe96 analyzer. The NSCs were generated and propagated using the NN1 media (see Chap. 1) with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The dopaminergic (DA) neurons were generated from the NSCs as described in Chap. 7. NSCs or their differentiated progeny, DA neurons, astrocytes, and oligodendrocytes offer an authentic and reliable system for screening drugs that target mitochondrial dysfunction in the central nervous system (CNS). The protocol described can be used to investigate acute versus chronic effects of the compounds on oxygen consumption rate (OCR) and on extracellular acidification rates (ECAR) of the cells. Furthermore the OCR and ECAR values can be used to calculate additional parameters of mitochondrial bioenergetics.

2 2.1

Materials Equipment

1. Seahorse XFe96 analyzer (Agilent, Seahorse Bioscience, North Billerica, MA, USA). 2. Seahorse XFe96 cell culture microplates (Agilent, Seahorse Bioscience, North Billerica, MA, USA). 3. Seahorse XFe96 sensor cartridges (Agilent, Seahorse Bioscience, North Billerica, MA, USA). 4. Hemocytometer (Hausser Scientific, Horsham, PA, USA) or automated cell counter (Countess, Invitrogen, Carlsbad, CA, USA). 5. Cell culture incubator (Nuaire, Plymouth, MN, USA). 6. 37  C non-CO2 incubator (Fisher Scientific, Pittsburgh, PA, USA). 7. Phase contrast microscope (Zeiss, Oberkochen, Germany). 8. pH meter (Corning/Fisher Scientific, Pittsburgh, PA, USA).

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9. Centrifuge tubes (15–50 mL) (Corning, Oneonta NY, USA). 10. Syringe filters (Corning, Oneonta NY, USA). 11. 60 mL syringes (BD, Franklin Lakes, NJ, USA). 12. Centrifuge (Eppendorf, Hamburg, Germany). 13. Multichannel pipettes (20–200 μL) (Eppendorf). 14. Reservoir basins (Fisher Scientific, Pittsburgh, PA, USA). 15. Pipette aids (10–1000 μL) (Eppendorf, Hamburg, Germany). 16. Pipette tips (10–1000 μL) (Accuflow, E&K Scientific, Santa Clara, CA, USA). 17. Water bath (Fisher Scientific, Pittsburgh, PA, USA). 2.2

Reagents

1. XF calibrant (Agilent, Seahorse Bioscience, North Billerica, MA, USA). 2. Seahorse XF Cell Mito Stress Test Kit (Agilent, Seahorse Bioscience, North Billerica, MA, USA): (a) Oligomycin. (b) Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). (c) Rotenone. 3. NN1 media (Neoneuron, San Antonio, TX, USA). 4. Fetal bovine serum (FBS, GE, Logan, Utah, USA). 5. Accutase (Gibco, Life Technologies, NY, USA). 6. Trypsin neutralizer (Gibco, Life Technologies, NY, USA). 7. Poly-L-ornithine hydrobromide (Sigma-Aldrich, St. Louis, MO, USA). 8. Sterile phosphate buffered solution (PBS, Gibco, Life Technologies, NY, USA). 9. Double-distilled water (Gibco, Life Technologies, NY, USA). 10. Artificial cerebrospinal fluid solution (ACSF): (a) Sodium chloride (NaCl) (Sigma-Aldrich, St. Louis, MO, USA). (b) Potassium chloride (KCl) (Sigma-Aldrich, St. Louis, MO, USA). (c) Calcium chloride (CaCl2) (Sigma-Aldrich, St. Louis, MO, USA). (d) Monopotassium phosphate (KH2PO4) (Sigma-Aldrich, St. Louis, MO, USA). (e) Magnesium chloride (MgCl2) (Sigma-Aldrich, St. Louis, MO, USA). (f) HEPES (Sigma-Aldrich, St. Louis, MO, USA). (g) Glucose (Sigma-Aldrich, St. Louis, MO, USA).

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(h) Sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA). (i) Sodium hydroxide (NaOH) (Sigma-Aldrich, St. Louis, MO, USA). (j) Hydrochloric acid (HCl) (Sigma-Aldrich, St. Louis, MO, USA).

3

Methods

3.1 Preparation of Reagents

1. ACSF: To prepare 100 mL of ACSF, dissolve the reagents in 90 mL of double-distilled (D.D, 18 MΩ cm at 25  C) (see Note 1) water to attain the following final concentrations. 120 mM NaCl, 3.5 mM KCl, 1.3 mM CaCl2, 0.4 mM KH2PO4, 1 mM MgCl2, 5 mM HEPES, 10 mM glucose, and 10 mM sodium pyruvate. Adjust the pH to 7.4 using either 1 N NaOH or 1 N HCL. Increase the volume to 100 mL, and recheck the pH. Filter sterilize and store at 4  C (see Notes 2 and 3). 2. Poly-L-ornithine hydrobromide (PLO): Dissolve poly-L-ornithine hydrobromide (PLO) in D.D water under sterile conditions to prepare a 10X stock solution of 0.16 mg/mL. The stock solution can be aliquoted and stored at 20  C for future use (see Note 1). 3. Culture media: To 50 mL of NN1 media, add 500 μL fetal bovine serum (final concentration 1%) to prepare culture media for plating NSCs. Filter sterilize and store at 4  C.

3.2 Coating XFe96 Cell Culture Microplates

1. Under sterile cell culture conditions, prepare working solution of PLO (16 μg/mL) by diluting 1 mL of 10 stock solution (0.16 mg/mL) in 9 mL of D.D water. 2. Add 25–30 μL of PLO working solution to each well, and incubate in cell culture incubator for a minimum of 2 h. 3. After incubation, wash the plate twice with sterile PBS and plate the cells. The plates can be prepared a day in advance and stored at 4  C under sterile conditions.

3.3 Plating Cells in Seahorse XFe96 Analyzer Cell Culture Microplates

(For the isolation and culture of iPSC-derived NSCs, please refer to Chap. 1. For the differentiation of NSCs to DA neurons, please refer to Chap. 7). In this protocol the cells should be plated in the XFe96 cell culture microplates a day before the actual assay. The workflow of the assay is outlined in Fig. 1. 1. Prewarm the Accutase, trypsin neutralizer, and culture media (NN1 + 1% FBS) in 37  C water bath. 2. Collect all the neurospheres from the T-75 flask into a 50 mL tube, and centrifuge at 1000 rpm (200  g) for 5 min.

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Fig. 1 Workflow for screening mitoeffective compounds using iPSC-derived dopaminergic neurons and XFe96 analyzer. Generate iPSCs from patient somatic cells and isolate NSCs. Culture and differentiate the NSCs into dopaminergic neurons in cell culture plate of XFe96 analyzer. Treat (acute or chronic) the dopaminergic neurons with small molecule library. Measure changes in OCR using XFe96 analyzer. Evaluate the effect of the test compounds on different components of mitochondrial respiration. Select potential compounds for further testing from the pool based on their efficacy on improving mitochondrial respiration

3. Carefully remove the supernatant by using vacuum suction without disturbing the pellet. 4. Resuspend the pellet in 1 mL Accutase, and incubate for 3 min at 37  C. 5. Inhibit the action of Accutase by adding 1 mL of trypsin neutralizer, and gently triturate 8–10 times to dissociate the cells into single-cell suspension. The trituration should be gentle enough to dissociate the cells without damaging the cellular integrity (see Note 4). 6. Add 8 mL of culture media, and centrifuge at 1000 rpm (200  g) for 5 min. 7. Remove the supernatant, and resuspend the cells in 3–5 mL of culture media. 8. Count the cells using either hemocytometer or automate cell counter, and prepare a cell suspension of 500,000 cells/mL (see Note 5). 9. Using a multichannel pipette, add 100 μL of cell suspension (50,000 cells/well) to all wells except A1, A12, H1, and H12 (Fig. 2). Add only media to A1, A12, H1, and H12 wells, as they will serve as blanks during the assay for background correction. 10. Incubate the cells for 1 h at 37  C in CO2 incubator. 11. To study long-term (24-h) effect of compounds on mitochondrial functions, prepare the compounds in 2 concentration in culture media and add to wells (100 μL/well). For control wells add 100 μL of culture media (see Note 6). 12. Incubate the cells at 37  C in CO2 incubator. 13. The following day check the morphology of the cells under a phase contrast microscope. If the cell density and plating was done properly, all the cells should attach and appear as a monolayer. If large numbers of vacant spaces are visible between the cells, then the plating density should be increased until a monolayer is observed.

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Fig. 2 Cell culture plate design of XFe96 analyzer. The protocol for XFe96 analyzer necessitates leaving four wells (depicted in black color: A1, A12, H1, and H12) blank for the measurement of background during the assay. The remaining 92 wells are used for plating cells and screening compounds 3.4 Hydrating Seahorse XFe96 Analyzer Sensor Cartridge

Hydrate the Seahorse XFe96 analyzer sensor cartridge at least for 12–24 h before the start of the assay; 1. Place the cartridge container label side up, and open the packaging. 2. Carefully lift the cartridge sensor along with the hydration plate, and place it on a level surface. The sensor cartridges come with two delivery guides that are helpful for loading compounds into the port. 3. Carefully place the sensor cartridges along with lid and delivery guides upside down. The probes of the sensor cartridge should not touch any kind of surface. 4. Add 200 μL of calibrant solution to each well of the hydration plate. 5. Place the sensor cartridge back on to the hydration plate making sure all the probes are in the calibrant solution. 6. Wrap the sensor cartridge in plastic, and place it overnight at 37  C in a non-CO2 incubator. 7. Turn on the XFe96 analyzer, and let it warm overnight.

3.5 Preparation of Cell Culture Microplate and Sensor Cartridge Plate for the Assay

The following steps are performed on the day of the assay: 1. Prewarm the ACSF in a 37  C water bath. 2. Check the cells under a phase contrast microscope for their morphology. The cells should appear as a monolayer with clear media without any floating cells. If large vacant spaces are

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visible between the cells, then the plating number should be adjusted until a monolayer is formed. 3. Using a multichannel pipette, carefully remove 180 μL of the media leaving 20 μL in each well (including blanks). Care should be taken to not disturb the cells. 4. Wash the cells twice with 200 μL of ACSF. 5. After the second wash, add 160 μL of ACSF to all the wells. 6. Check the morphology of the cells under a phase contrast microscope to make sure the monolayer is intact in all wells and no cells are lost (see Note 7). 7. Place the cell culture microplate in a non-CO2 incubator at 37  C for the ACSF to equilibrate. 8. Meanwhile prepare stock solutions of the compounds for the assay in ACSF. 9. For mitochondrial modulators in the Mito Stress Kit, prepare the stock solution according to manufacturer’s instructions in ACSF (assay media): (a) Oligomycin: add 630 μL of ACSF and mix well, final concentration of 100 μM. (b) FCCP: add 720 μL of ACSF and mix well, final concentration of 100 μM. (c) Rotenone: add 540 μL of ACSF and mix well, final concentration of 50 μM (see Note 8). 10. Prepare 10 concentration working solutions of the mitochondrial modulators: (a) Oligomycin: add 600 μL of stock solution to 2.4 mL of ACSF, concentration of 20 μM (10). (b) FCCP: add 600 μL of stock solution to 2.4 mL of ACSF, concentration of 20 μM (10). (c) Rotenone: add 300 μL of stock solution to 2.7 mL of ACSF, concentration of 5 μM (10). 11. Prepare the test compounds for drug screening (acute response) at 10X concentration in ACSF. Calculate the required volume for each drug by multiplying the number of replicates with 20 μL/well. 12. Load the compounds using delivery guide into the ports of the sensor cartridge. Do not remove the hydration plate during this process. The probes should always remain hydrated during the entire procedure. 13. For experiments investigating chronic response to compounds on mitochondrial respiration, load the compounds in the following order:

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Concentration (μM) Port

Compound

Volume (μL)

Port

Well

A

Oligomycin

20

20

2

B

FCCP

22

20

2

C

Rotenone

25

5

0.5

14. For experiments investigating acute response to compounds on mitochondrial respiration, load the compounds in the following order: Concentration (μM) Port

Compound

Volume (μL)

Port

Well

A

Test Compound

20

10



B

Oligomycin

20

20

2

C

FCCP

22

20

2

D

Rotenone

25

5

0.5

15. Place the pipette tip through the holes in the delivery guide into each port, and carefully dispense the solution. 16. Ensure all the ports are filled with their designated compounds. 3.6 Real-Time Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR)

In this protocol, the XFe96 analyzer should be turned on the evening before the day of the assay: 1. Initiate the seahorse wave software program. 2. Select the Mito Stress template. 3. Enter the information regarding the experiment, i.e., cell type, cell number, assay media treatment, date, etc. 4. Select the wells and label them accordingly (blanks, control, and treatment). 5. Label the injection ports with their respective compounds. 6. Enter the measurement settings: Calibrate. Equilibrate. Baseline: Mix: 3 min. Wait: 1 min. Measure: 3 min. Loop 3 times.

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Injection 1: Oligomycin. Mix: 3 min. Wait: 1 min. Measure: 3 min. Loop 3 times. Injection 2: FCCP. Mix: 3 min. Wait: 1 min. Measure: 3 min. Loop 3 times. Injection 3: Rotenone. Mix: 3 min. Wait: 1 min. Measure: 3 min. Loop 3 times. 7. For experiments investigating the acute response of the investigational compound, the following settings can be used: Calibrate. Equilibrate. Baseline: Mix: 3 min. Wait: 1 min. Measure: 3 min. Loop 3 times. Injection 1: Test compound. Mix: 3 min. Wait: 1 min. Measure: 3 min. Loop 3 times. Injection 2: Oligomycin. Mix: 3 min. Wait: 1 min. Measure: 3 min. Loop 3 times. Injection 3: FCCP. Mix: 3 min. Wait: 1 min. Measure: 3 min.

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Loop 3 times. Injection 4: Rotenone. Mix: 3 min. Wait: 1 min. Measure: 3 min. Loop 3 times. 8. Proceed to next step. When prompted by the software, remove the lid and load the sensor cartridge along with the hydration plate into the slot provided in the machine. Make sure the sensor cartridges are placed in proper orientation and in alignment with the slot of the machine. 9. Begin the calibration process. 10. Once the calibration is completed, the software will prompt to load the cell culture microplate. Replace the hydration plate with the cell culture microplate. Make sure the cell culture microplate is in proper orientation and in alignment with the slot of the machine. 11. Start the assay. 12. After completion of the assay, save the results file and export results to an excel document. 13. Save the plate for further analysis, such as protein estimation. 3.7

Data Analysis

1. The excel document provides detailed information regarding assay configuration, raw values, rate, and operation log. 2. The raw (plate) values provide information of each measurement and can be used to plot the changes in OCR or ECAR in response to the compounds used in the test against time. 3. In this protocol using the OCR values from the assay results, the following additional parameters can be calculated (see Note 9): (a) Non-mitochondrial respiration: third OCR measurement after rotenone injection. (b) Basal respiration: (third baseline OCR measurement) – non-mitochondrial respiration. (c) Maximal respiration: (Highest OCR measurement after the injection of FCCP)—(non-mitochondrial respiration). (d) ATP-linked respiration: (third baseline OCR measurement)—(third measurement following oligomycin injection). (e) Proton leak: (third measurement following oligomycin injection)—non-mitochondrial respiration.

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Fig. 3 Measurement of changes in OCR in response to treatment with different compounds. Representative plots demonstrating the changes in OCR in response to treatment with different test compounds. Each compound was tested in three different concentrations to determine the differential response of drug concentration on cellular OCR

(f) Spare respiratory capacity: Maximal respiration—basal respiration. 4. The data can be presented as absolute OCR (Mmol/min) (Fig. 3) and ECAR (pH/min) values or normalized to the cell number or protein content of the wells.

4

Notes 1. Prepare all solutions and reagents in double-distilled water or ultrapure water (18 MΩ cm at 25  C). 2. In contrast to other protocols that use XF assay medium, we used ACSF in our assays. We have compared the results from XF assay medium and ACSF and observed that NSCs survived and responded better in ACSF compared to XF assay media. Furthermore, ACSF is recommended for the assays with primary rat cortical neurons.

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3. Adjust the pH of ACSF accurately to 7.4. The assay is sensitive to changes in pH, and this may affect the outcome of the tests. 4. During the dissociation of neurospheres to generate single-cell suspension, the trituration should be done gently to reduce damage to the integrity of the cells but should also be firmly enough to dissociate larger clumps as they may affect the plating density and thus the results of the assay. 5. A cell density titration should be performed to determine the cell number for optimal detection of response in the assay. Usually a cell density of 30,000–50,000 cells/well should be sufficient to generate a discernable response in the assay. However, it may change according to the experiment parameters (cell type, treatment). 6. It is recommended to have at least 4–6 replicates per group to obtain accurate results in the assay. 7. Care should be taken during the rinsing process as loss of cells results in high variability between and within groups. 8. Rotenone is toxic and known to cause Parkinson’s disease. Personal protective equipment should be worn all the time while working with rotenone and other toxic compounds. 9. The calculations are based on the experimental setup described in the current protocol. Changes in the number of measurements may require modification to the equations for the calculation of the parameters.

Acknowledgment The authors thank the members of the Daadi laboratory for their helpful support and suggestions. This work was supported by the Worth Family Fund, the Perry & Ruby Stevens Charitable Foundation, the Robert J., Jr. and Helen C. Kleberg Foundation, the NIH primate center base grant (Office of Research Infrastructure Programs/OD P51 OD011133), and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1 TR001120. Disclosures: Dr. Marcel M. Daadi is founder of the biotech company NeoNeuron. References 1. Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443(7113):787–795 2. Zhang J et al (2012) Measuring energy metabolism in cultured cells, including human

pluripotent stem cells and differentiated cells. Nat Protoc 7(6):1068–1085 3. Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435(2):297–312

Dopamine Neurons Mitochondrial Assay 4. Wang R et al (2015) The acute extracellular flux (XF) assay to assess compound effects on mitochondrial function. J Biomol Screen 20 (3):422–429 5. Koopman M et al (2016) A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans. Nat Protoc 11 (10):1798–1816 6. Ozsvari B et al (2017) Mitoriboscins: Mitochondrial-based therapeutics targeting cancer stem cells (CSCs), bacteria and pathogenic yeast. Oncotarget 8(40):67457–67472 7. Ribeiro SM, Gimenez-Cassina A, Danial NN (2015) Measurement of mitochondrial oxygen consumption rates in mouse primary neurons and astrocytes. Methods Mol Biol 1241:59–69 8. Clerc P, Polster BM (2012) Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons. PLoS One 7(4):e34465 9. Jiang T, Cadenas E (2014) Astrocytic metabolic and inflammatory changes as a function of age. Aging Cell 13(6):1059–1067

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10. Roy Choudhury G et al (2015) Methylene blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration. PLoS One 10(4):e0123096 11. Park SJ et al (2017) Metabolome profiling of partial and fully reprogrammed induced pluripotent stem cells. Stem Cells Dev 26 (10):734–742 12. Panopoulos AD et al (2012) The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming. Cell Res 22(1):168–177 13. Rana P et al (2012) Characterization of human-induced pluripotent stem cell-derived cardiomyocytes: bioenergetics and utilization in safety screening. Toxicol Sci 130 (1):117–131 14. van der Windt GJ, Chang CH, Pearce EL (2016) Measuring bioenergetics in T cells using a seahorse extracellular flux analyzer. Curr Protoc Immunol 113:3 16B 1–3 16B 14 15. Dong Z et al (2016) Focused screening of mitochondrial metabolism reveals a crucial role for a tumor suppressor Hbp1 in ovarian reserve. Cell Death Differ 23(10):1602–1614

Chapter 13 Reference Transcriptome for Deriving Marmoset Induced Pluripotent Stem Cells Guang Yang, Hyenjong Hong, April Torres, Kristen E. Malloy, Gourav Roy-Choudhury, Jeffrey Kim, and Marcel M. Daadi Abstract Limited access to primary tissue from various nonhuman primate (NHP) species represents a significant unmet need that hampers progress in understanding unique cellular diversity and gene regulation of specific tissues and organs in stem cell translational research. Most comparative biology studies have been limited to using postmortem tissue usually frozen specimens with limited utility for research. The generation of induced pluripotent stem cell (iPSC) lines from somatic cells, such as adult skin or blood cells, offers an alternative to invasive and ethically controversial interventions for acquiring tissue. Pluripotent iPSCs have virtually an unlimited capacity to proliferate and differentiate into all cell types of the body. We are generating high-quality validated NHP iPSC lines to offer to scientific community and facilitate their research programs. We use the non-integrative episomal vector system to generate iPSCs from NHP skin biopsies. In this chapter we describe the validation of NHP iPSC lines by confirming pluripotency and their propensity to differentiate into all three germ layers ectoderm, mesoderm, and endoderm according to established standards and measurable limits for a set of marker genes incorporated into a scorecard. Key words Marmoset iPSCs, Pluripotency, Scorecard, iPSC characterization, Nonhuman primate iPSCs, Regenerative medicine

1

Introduction Regenerative medicine focuses on creating vital functional cells to repair or replace tissue or organs damaged due to disease, injury, or congenital defects. This approach may provide treatments for currently intractable diseases. The burgeoning of this medical approach has been galvanized by the potential of pluripotent stem cells (iPSCs). The possibility to generate specialized organ-specific differentiated cells using human somatic cells induced to become iPSCs opens the door for individualized cell therapy. Individualized autologous or allogeneic cell therapy would be ideal for treating various diseases or injuries such as heart attacks, diabetes, stroke, neurodegenerative diseases, and others. Furthermore, current

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_13, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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limited access to primary tissue from various parts of the body represents a significant unmet need that hampers progress in understanding the unique cellular diversity and gene regulation of specific tissues and organs and in stem cell translational research. Most comparative studies have been limited to using postmortem tissue usually frozen, specimens with limited utility for research. Pluripotent iPSCs have virtually an unlimited capacity to proliferate and differentiate into all cell types of the body. Thus, the generation of iPSC lines will obviate the need for invasive interventions to acquire primary tissue and for the use of frozen tissues. Animal models have historically made a significant contribution to our understanding of human diseases. However, disparities between results in animal studies and clinical trials have been identified, including failure to acknowledge the limitations of animal species and disease models [1]. Measuring the therapeutic potential of stem cell lines through testing in small and large animal models may predict the safety and efficacy of treatment strategies for clinical trials. NHP provide important disease models for translational regenerative medicine [2]. Given the similarities to humans, in anatomical structure, physiology, and pathology, NHP are also relevant in comparative biology and medicine. The creation of a resource of standardized, high-quality and validated nonhuman primates (NHP) iPSC lines will offer access to efficient and reliable model systems of all cell types of the body. This resource facilitates research and development in cell therapy, disease modeling, drug screening, and comparative functional genomics and medicine. Here, we describe methods and standards we use in our lab to derive defined pluripotent NHP iPSCs.

2 2.1

Materials Equipment

1. Cell culture incubator (Nuaire, Plymouth, MN, USA). 2. Phase contrast microscope (Zeiss, Oberkochen, Germany). 3. StepOnePlus (Applied Biosystems, Waltham, MA, USA). 4. TaqMan hPSC Scorecard Kit, Fast 96-well (Applied Biosystems, Waltham, MA, USA). 5. MicroAmp Optical Adhesive Film (Applied Biosystems, Waltham, MA, USA). 6. Centrifuge (Eppendorf, Hamburg, Germany). 7. 6-, 24-, 48-, and 96-well cell culture plates (Corning, Oneonta, NY, USA). 8. Corning 6-well Clear Flat Bottom Ultra Low Attachment Multiple Well Plates (Corning, Oneonta, NY, USA). 9. 60 mm petri dish (BD biosciences, Franklin Lakes, NJ, USA).

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10. Cell lifter (Fisher scientific, Pittsburgh, PA, USA). 11. Glass pipettes (Fisher scientific, Pittsburgh, PA, USA). 12. Centrifuge tubes (15 mL and 50 mL) (Corning, Oneonta, NY, USA). 13. Syringe filters (Corning, Oneonta, NY, USA). 14. Syringes (10, 20 and 60 mL) (BD, Franklin Lakes, NJ, USA). 15. Pipettes (2–25 mL) (Fisher scientific, Pittsburgh, PA, USA). 16. Pipette aids (10–1000 μL) (Eppendorf, Hamburg, Germany). 17. Pipette tips (10–1000 μL) (Accuflow, E&K Scientific, Santa Clara, CA, USA). 18. Water bath (Fisher scientific, Pittsburgh, PA, USA). 19. Hemocytometer (Hausser Scientific, Horsham, PA, USA) or automated cell counter (Countess, Invitrogen, Carlsbad, CA, USA). 2.2 Kits and Reagents

1. Episomal plasmids (pCXLE-hOCT3/4-shp53-F, pCXLEhSK, pCXLE-hUL, and pCXWB-EBNA1, Addgene, Cambridge, MA, USA). 2. NHDF Nucleofector Kit (Lonza, Walkersville, MD, USA). 3. Amaxa Nucleofector device 2b (Lonza, Walkersville, MD, USA). 4. Mycoplasma PCR detection kit (Sigma-Aldrich, St. Louis, MO, USA). 5. DMEM (Gibco, Life Technologies, NY, USA). 6. DMDM/F12 (Gibco, Life Technologies, NY, USA). 7. RNeasy Plus Mini kit (QIAGEN, Germantown, MD, USA). 8. Irradiated mouse embryonic fibroblasts. 9. KnockOut Serum Replacement (Gibco, Life Technologies, NY, USA). 10. L-Glutamine (Gibco, Life Technologies, NY, USA). 11. MEM Non-Essential Amino Acids Solution (Gibco, Life Technologies, NY, USA). 12. Penicillin-streptomycin (Gibco, Life Technologies, NY, USA). 13. β-Mercaptoethanol (Gibco, Life Technologies, NY, USA). 14. Basic fibroblast growth factor (Stemgent, Cambridge, MA, USA). 15. Fetal bovine serum (FBS, GE, Logan, Utah, USA). 16. Gelatin (Sigma-Aldrich, St. Louis, MO, USA). 17. Collagenase Type IV solution (Sigma-Aldrich, St. Louis, MO, USA).

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18. Trypsin (Gibco, Life Technologies, NY, USA). 19. Trypsin neutralizer (Gibco, Life Technologies, NY, USA). 20. TaqMan hPSC Scorecard Kit, Fast 96-well (Applied Biosystems, Waltham, MA, USA). 21. SuperScript IV first-strand synthesis system (Invitrogen, Thermo Fisher, Waltham, MA, USA). 22. TaqMan Fast Advanced Master Mix (Applied Biosystems, Waltham, MA, USA). 23. Tris base (Fisher Bioreagents, Pittsburgh, PA, USA). 24. Phosphate buffered saline (PBS, Gibco, Life Technologies, NY, USA). 25. Double distilled water (Gibco, Life Technologies, NY, USA). 26. Ethanol (Fisher Bioreagents, Pittsburgh, PA, USA).

3

Methods

3.1 Preparation of Reagents and Media: Prepare All the Reagents Under Sterile Conditions in a Horizontal Laminar Flow Hood

1. Preparation of 0.1% gelatin: Dissolve 0.5 g gelatin in 500 mL ddH2O. Autoclave (20 min at 120
C) and store at room temperature under sterile conditions. 2. Skin biopsy media (50 mL): Mix 40 mL of DMEM, 10 mL of fetal bovine serum (20%), and penicillin/streptomycin (1%). Filter sterilize and store at 4
C. 3. Fibroblast media (50 mL): Mix 45 mL of DMEM, 5 mL of fetal bovine serum (10%). Filter (2 μm) sterilize and store at 4
C. 4. Preparation of 10 mM Tris (25 mL): Dissolve 30.35 milligrams of Tris base (F.Wt, 121.4) in 15 mL of double-distilled water and adjust the pH to 7.6. Increase the volume to 25 mL and filter sterilize. Store at 4
C. 5. Preparation of basic fibroblast growth factor (bFGF) stock solution: Briefly centrifuge the tube and reconstitute the bFGF (50 μg) in 2.5 mL of 10 mM Tris solution (pH 7.6) to prepare a 20 μg/mL stock solution. Aliquot and store at 20
C. 6. Preparation of human ESC (hESC) media (250 mL): Mix 196.25 mL of DMDM/F12, 50 mL of KnockOut Serum Replacement (20%), 2.5 mL of 10 mM MEM Non-Essential Amino Acids Solution, 1.25 mL of L-glutamine with 1.75 μL of β-mercaptoethanol, and 125 μL of 20 μg/mL bFGF (final 10 ng/mL). Filter sterilize and store at 4
C. 7. Preparation of Embryoid Body (EB) media (50 mL): Mix 40 mL of DMEM/F12, 10 mL KnockOut Serum Replacement (20%), 1 mM MEM Non-Essential Amino Acids Solution, and 55 μM β-mercaptoethanol. Filter sterilize and store at 4
C.

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Fig. 1 Process of generation, characterization, and validation of iPSC lines. Go/No-Go Workflow to generate, characterize and bank NHP iPSC lines. Adapted from [3]

8. Preparation of Collagenase Type IV solution: Dissolve 1 mg to 1 mL of ddH2O and and filter sterilize. Aliquot and store at 20
C. 3.2 Generation of Marmoset iPS Cells (CJ-iPSCs) from Skin Biopsy (See Note 1) (Fig. 1)

1. Coat the 6-well cell culture plate with 0.1% gelatin solution (1 mL per well) and incubate in 37
C cell culture incubator for a minimum of 30 min. 2. Select 4- to 8-year-old healthy marmosets (Callithrix Jacchus) and take skin biopsies (4 to 6 mm) from a preferably hairless regions like abdomen or underarm (see Note 2). 3. Transport the skin biopsies in sterile PBS containing 1% penicillin and streptomycin to the cell culture facility. The following steps are performed in the cell culture facility in a horizontal laminar flow hood under sterile conditions. 4. Using a sterile surgical blade, mince each skin piece into 1 mm or less pieces in a 60 mm petri dish.

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5. Aspirate the gelatin solution from the culture plate using vacuum suction. 6. Using sterile forceps, carefully transfer the tissue pieces into the gelatin-coated wells. Typically 4–5 pieces of minced skin tissue can be placed into a single well of 6-well culture plate. 7. Carefully add 0.8 mL of skin biopsy media along the wall of the well without displacing the tissue pieces. 8. Incubate the plate at 37
C incubator for 2 days. Do not move the culture plates until the biopsy pieces have attached to the bottom of wells. 9. After 2 days, add 0.2 mL of fresh media to the wells without removing the old media. Repeat the step every 2 days. 10. After 1 week, fibroblasts can be seen growing from the biopsy tissue. 11. Change media regularly until the fibroblasts become confluent. 12. When the fibroblasts become confluent, expand the cells to a T-25 cell culture flask. 13. Perform mycoplasma test using Hoechst 33258 DNA staining and mycoplasma PCR detection Kit. 14. Expand the fibroblasts to 2–5 passages for freezing stocks and for reprogramming. 15. The day before transfection, coat a 10 cm cell culture dish with gelatin and plate irradiated mouse embryonic fibroblasts (MEF) feeder with fibroblast media. 16. On the day of transfection, change the media of irradiated MEF feeder with fresh fibroblast media. All the following steps should be performed on a bench that has been cleaned and sterilized with 70% ethanol. 17. Set up NHDF Nucleofector kit on a clean bench (see Note 3). 18. Mix 82 μL supplement (0.83 μg of hSK, 0.83 EBNA1).

of NHDF Nucleofector solution with 18 μL of and add 3 μg of human episomal plasmids pCXLE-hOCT3/4-shp53-F, 0.83 μg of pCXLEμg of pCXLE-hUL, and 0.5 μg of pCXWB-

19. Dissociate the marmoset fibroblasts with trypsin (passage number 3 to 6), and determine the cell density with cell counter or hemocytometer. 20. Prepare a cell suspension of 500,000 fibroblasts and centrifuge at 1000 rpm (200  g) for 5 min. 21. Remove the supernatant, and resuspend the cell pellet with Nucleofector solution-plasmids mixture.

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22. Transfer cell-DNA mixture into a Nucleofector cuvette. Take care to prevent the formation of bubbles in the solution during transfer. 23. Load the cuvette into the Nucleofector device and start the program U-023. 24. After the completion of nucleofection, immediately transfer the cells onto irradiated MEF feeder and culture for 3 days. 25. On day 3 after transfection, replace the fibroblast media with fresh hESC media. 26. Replace old media with fresh hESC media every other day. 27. Culture the cells for 5 weeks or until hESC-like colonies emerge. 28. When the colonies emerge, prepare irradiated MEF feeder in a 24-well plate 1 day before colony picking. 29. On the day of colony picking, set up the microscope on a clean bench. 30. Change the media of irradiated MEF feeder with fresh hESC media. 31. Prepare a 96-well plate with 20 μL of trypsin per well. 32. Using P10 pipettor, pick the colonies and transfer them to trypsin-containing wells (see Note 4). 33. Incubate the colony-loaded 96-well plate at 37
C for 5 min. 34. Add 150 μL of hESC media to each well and dissociate the colonies by pipetting 30 times. 35. Plate the dissociated cells on the irradiated MEF feeder in the 24-well plate. 36. Change the media daily with hESC media until the CJ-iPSCs become confluent. 37. Expand the CJ-iPSCs to 6-well plates to scale up and to prepare frozen stocks. 3.3 Embryoid Body (EB) Formation

1. Culture marmoset iPSCs (CJ-iPSCs) with irradiated MEF feeder layers in 6-well plate until they are confluent (Fig. 2). 2. Wash the cells with sterile DPBS and add 0.5 mL of collagenase type IV, and incubate at 37
C for 5 min to remove the irradiated MEFs. 3. During incubation, check the cells every 2 min under the microscope to gauge the level of MEF detachment. 4. When the majority of the MEFs are detached (the marmoset iPSCs should not detach), proceed to the next step. 5. Aspirate collagenase-MEF supernatant and wash with sterile DPBS taking care not to detach marmoset iPSCs.

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Fig. 2 IPSC colonies derived by reprograming skin fibroblast using the episomal approach. (a) Phase contrast photo showing example of two marmoset iPSC colonies. (b) Marmoset iPSC colonies express the pluripotent marker Nanog. Scale bar: 100 μm

6. Add 1 mL of fresh hESC media, and detach all the marmoset iPSC colonies using a cell lifter. 7. Transfer the cell suspension into two 15 mL tubes and centrifuge at 200  g for 5 min. 8. Aspirate the supernatant carefully and store (short-term) the cells from one tube at 80
C for RNA isolation (undifferentiated iPSCs). 9. Add 2 mL of pre-warmed EB media to the other tube and gently resuspend the colonies while taking care not to dissociate them. 10. Transfer the cell suspension into a 6-well ultra-low attachment plate and culture for 7 days with media changed every other day with fresh EB media. 11. To change media, transfer the cell suspension into a 15 mL tube and allow the EBs to settle at the bottom of tube under gravity (for 5 min). 12. Carefully remove the supernatant and resuspend with fresh media. 13. On day 7, collect all the EBs by centrifugation (200  g 5 min). 3.4 RNA Isolation Using RNeasy Plus Mini Kit (Fig. 3)

1. Thaw the frozen undifferentiated iPSCs stored at 80
C. 2. Prepare 1 mL of Buffer RLT and add 10 μL of β-mercaptoethanol. 3. Resuspend the CJ-IPSC pellet with 350 μL of RLTβ-mercaptoethanol mixture and triturate to lyse all the cells in the pellet. 4. Load the cell lysate into a gDNA eliminator spin columns and centrifuge at 13k rpm for 1 min and collect the flow-through.

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Fig. 3 Workflow of the scorecard: Representation of the steps leading to the pluripotency and differentiation standard tests—left the pluripotency and right the EB differentiation standard test. Adapted from [3]

5. Add 350 μL of 70% ethanol to the eluate and load the mixture into RNeasy spin column and centrifuge at 13k rpm for 1 min. Discard the flow-through. 6. Add 700 μL of RW1 and spin down 13k rpm for 1 min. Discard the flow-through. 7. Add 500 μL of RPE and spin down 13k rpm for 1 min. Discard the flow-through. Repeat this process. 8. Replace the column on the new 1.5 mL tube. Expel 30 μL of RNase-free water on the center of membrane in column. Hold for 1 min. 9. Centrifuge at 13k rpm for 1 min and elute the RNA. 10. Measure the concentration of RNA. Use 1 μg of RNA to determine RNA degradation by gel electrophoresis.

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3.5 Complementary DNA Synthesis Using SuperScript IV FirstStrand Synthesis System

1. In a PCR tube, dilute 1 μg of RNA in 11 μL of RNase-free water. 2. Combine 1 μL of 50 μM Oligo d(T)20 with 1 μL of 10 mM dNTP mix. 3. Place the mixture at 65
C for 5 min followed by placement on ice for 1 min. 4. Add 4 μL of 5 SSIV buffer, 1 μL of 100 mM DTT, 1 μL of ribonuclease inhibitor, and 1 μL of SuperScript IV Reverse Transcriptase. 5. Place the mixture at 50
C for 10 min and then at 80
C for 10 min for inactivation.

3.6 Scorecard Panel (Fig. 4)

1. Add ddH2O to each cDNA sample to prepare a mixture with final volume 70 μL. 2. Add 70 μL of 2 TaqMan Fast Advanced Master Mix. 3. Load 10 μL into each well of one row of TaqMan hPSC Scorecard Kit, Fast 96-well. 4. Seal the plate using MicroAmp Optical Adhesive Film and spin down at 600  g for 2 min. 5. Load in StepOnePlus and run in Fast Run mode. Step

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6. Save the results as eds file. 7. Upload the files to the Thermo Fisher’s TaqMan hPSC Scorecard Panel and data analysis site. https://www.thermofisher.com/ us/en/home/life-science/stem-cell-research/taqman-hpsc-score card-panel/scorecard-software.html. 8. Sign in with user ID and password. 9. Click “Create an analysis group” and label the folder. 10. Select “StepOnePlus System” under Instrument Type and “96 Wells Fast” under Block and then click “OK.” 11. Click “Import Data” and load the eds files. 12. If done properly the message “Uploading to Thermo Fisher cloud successful” will appear. 13. Check the box on the left-hand side of each sample and then click “Plots” to compare expression, scores box plot, scores table, correlation, and assay QC.

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Fig. 4 The scorecard test. (a) Scorecard assay pass for 7-day-old embryoid bodies (EBs) derived from marmoset iPSC line (CJ01). The EBs downregulated pluripotent self-renewal genes (self, green) as shown with the negative () sign and significantly upregulated ectoderm, mesoderm, and endoderm genes (Ecto, Meso, and Endo, respectively) as shown with the (+) sign. (b) Overview of Scorecard test of our marmoset iPSC line (CJ01) showing reference genes up- or downregulated. CT control, self-renewal ¼ pluripotent, ED endoderm, MS mesondoderm, ME mesoderm, EC ectoderm genes. The figure shows all the pluripotency genes downregulated (in blue) and the ME, EC, and ED genes upregulated. Adapted from [3]

14. The results can be downloaded as JPG by clicking “Download as JPG” located at the right upper corner of the screen.

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Notes 1. All nonhuman primate procedures should be authorized by local and regional governmental authorities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC international). All procedures involving animals should be performed in compliance with the guide for care and use of laboratory animals regulated by the Office of Laboratory Animal Welfare (OLAW) at the National Institutes of Health. The experimental procedures

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should be approved by an Institutional Animal Care and Use Committee (IACUC). 2. The skin biopsies should be taken by the veterinary staff according to standard operating procedures (SOP). 3. All reagents should be prepared 30 min in advance to allow them to reach ambient temperature for use. 4. The colonies should be picked within 20 min to prevent evaporation and drying of trypsin from the wells. 5. It is recommended to use marmoset ESCs as positive control.

Acknowledgments The authors thank members of the Daadi laboratory for helpful support and suggestions. This work was supported by the Worth Family Fund, The Perry & Ruby Stevens Charitable Foundation, The Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation, the NIH primate center base grant (Office of Research Infrastructure Programs/OD P51 OD011133), and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1 TR001120. Disclosures: Dr. Marcel M. Daadi is founder of the biotech company NeoNeuron. References 1. STAIR (1999) Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke 30:2752–2758 2. Daadi MM, Barberi T, Shi Q, Lanford RE (2014) Nonhuman primate models in translational regenerative medicine. Stem Cells Dev 23(Suppl 1):83–87

3. Yang G, Hong H, Torres A, Malloy KE, Choudhury GR, Kim J, Daadi MM (2018) Standards for deriving nonhuman primate-induced pluripotent stem cells, neural stem cells and dopaminergic lineage. Int J Mol Sci 19 (9):2788 https://doi.org/10.3390/ ijms19092788

Chapter 14 Optimization of Differentiation of Nonhuman Primate Pluripotent Cells Using a Combinatorial Approach Steven L. Farnsworth, Zhifang Qiu, Anuja Mishra, and Peter J. Hornsby Abstract The directed differentiation of pluripotent stem cells to a desired lineage often involves complex and lengthy protocols. In order to study the requirements for differentiation in a systematic way, we present here methodology for an iterative approach using combinations of small molecules and biological factors. The factors are used in a cyclical process in which the best combination of factors and concentrations is selected in one round of testing, followed by a modification of the combination and subsequent rounds. While this may produce the desired differentiation in the cell population under study, it is also possible that other strategies may be needed to optimize the differentiation process. These strategies are described in this chapter. Key words Pluripotent stem cells, Nonhuman primates, Differentiation, Algorithms, Gene expression

1

Introduction There is an ongoing need to devise efficient protocols for the differentiation of pluripotent stem cells into defined lineages. This may be required for basic science studies or for translational and therapeutic investigations. The methodology described in this chapter comprises a combinatorial approach, using factors hypothesized or demonstrated to promote differentiation into a desired lineage, such as neural, cardiac, immune system, and so on. The approach taken here is to rationally combine selected compounds in protocols that take advantage both of the results of prior studies and also of the increasing knowledge of the intracellular signaling pathways that have been identified as being involved. The methodology requires the identification of suitable molecular markers needed for differentiation; these may represent various developmental stages in the specific lineage being studied. A major concern is that the adaptation of protocols across species (e.g., from humans to nonhuman primates, NHPs) requires many adjustments to the

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_14, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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concentrations of the factors being used and in the timing of their use in the protocol. This chapter outlines a general method for neural differentiation of pluripotent stem cells, with specific reference to an NHP stem cell line, marmoset iPS cells. Methods for the differentiation of pluripotent stem cells into defined lineages typically use smallmolecule inhibitors or agonists, combined with biological factors, in various combinations and concentrations, used for various periods of time and added in various orders. Such methods can become extremely complex and offer huge numbers of potential combinations (factors, concentrations, time, order of addition). A systematic approach to this challenge is needed, and this is the approach outlined in this chapter. In previous publications we demonstrated that such a combinatorial approach, using small molecules and biological factors, can assist in the rapid optimization of a differentiation protocol [1]. We use a cyclical approach in which systematic variations in the concentrations of several factors are used to select a “winning” combination in each cycle. This cyclical approach has been termed feedback system control [2]. The protocol described here uses a “hill-climbing” algorithm in which systematic variations in the combinations and concentrations of factors used move the cell population toward a maximum state of differentiation [1]. We assume that the degree of differentiation of the cells following combination treatments is evaluable as a single value, so permitting a combination of factors at specific concentrations to be declared the “winner” at each round (see Note 1). Here we present this approach, with further variations included as alternatives to the main “hill-climbing” cycle, as shown in Fig. 1.

2

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An appropriate pluripotent stem cell line: here we used marmoset iPS cells B8 as previously characterized [3].

2.2 Cell Culture Medium and Related Materials

E8 medium (STEMCELL Technologies). DMEM/F12 (Sigma). KnockOut Serum Replacement (KSR; Invitrogen). Fetal bovine serum (GlobalStem). Y-27632; inhibitor of Rho-associated, coiled-coil-containing protein kinase (ROCK) (Fisher Scientific). Accutase (BioExpress, Kaysville, UT).

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Alternative protocol if optimization has stalled: Test addition or removal of factors or test significance of hypothesized signalling pathways: Formulate new base combination

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Combinations tested in short iPS cell differentiation protocol Assess outcomes: Any better combinations?

Fig. 1 Cyclical combinatorial scheme for differentiation of pluripotent cells, with variations added to permit analysis of specific issues. See Introduction for details. For an example of the outcome of a single round of testing, see Fig. 2; for examples of the outcome of the alternative approaches, other than the main cycle, see Figs. 3 and 4 2.3 Small-Molecule Differentiation Factors

Y-27632. Dorsomorphin (Fisher Scientific); a selective small-molecule inhibitor of BMP signaling. SB431542 (Selleckchem); inhibitor of the TGF-β/activin/nodal pathway that inhibits ALK5, ALK4, and ALK7. PD325901 (Biotang); inhibitor of MEK 1 and 2. BIO (Enzo); GSK-3 inhibitor.

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IWR-1 (Sigma); Wnt inhibitor, promotes β-catenin destruction. DAPT (Tocris); Notch inhibitor. All-trans retinoic acid (Sigma). BMP4 ligand (Peprotech). TGF-β1 (R&D Systems). FGF2 (basic FGF) (Stemgent). 2.4 Materials for Analysis by qPCR

ABI Prism 7900 HT Sequence Detection System (Applied Biosystems; ABI) and SDS analysis software. RNA-Bee (Tel-test). Superscript II reverse transcriptase kit (Invitrogen). Random primers (Promega). SYBR green (ABI). Primers: designed using Primer Express 2.0 software (ABI) against predicted mRNA sequence for marmoset based upon a sequence comparison between the marmoset genome (UCSC Genome Browser) and human Refseq mRNA; β-actin (ACTB) primers as reference gene.

3

Methods 1. Select an appropriate pluripotent stem cell line for these studies. Here we illustrate the methodology using a marmoset iPS cell line, B8, which we have previously characterized [3]. Propagate the cells under conditions that maintain the pluripotent characteristics of the cells (e.g., E8 medium plus 10% fetal bovine serum; ref. 4); this will be specific for the cell line chosen. 2. In the case of B8 marmoset iPS cells, begin by removing the cells from the culture surface with Accutase, and place the cells in nonadhesive U-bottom 96-well plates to create cell aggregates [1]. Use DME/F12 with 20% KSR [1]. This was found to be optimal for this cell line, but other cell lines may require different protocols (see Note 2). 3. After 24 h, change the medium on the cell aggregates to a basal differentiation medium (DME/F12 with 20% KSR) to which is added various combinations of factors to be tested. The table in Fig. 2 shows the combinations of factors used, based on prior results obtained with this cell line [1]. Adjust the combinations of factors used based on what is already known for the cell line under study (see Note 3).

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Fig. 2 Example of the results of one round of testing of combinations of various factors on differentiation of marmoset iPS cells. The concentrations shown in red in the table in the upper left were used in this round; other concentrations (initially level “3”) were used in prior rounds or may be used in future rounds. Only two concentrations of dorsomorphin were used because the higher concentrations were toxic. Expression of the neuroectodermal marker genes SOX1 and NCAD, and the neural crest markers SOX10 and TFAP2A, was assessed. The y axis represents the mRNA level as Ct(gene)-Ct(β-actin); higher bars indicate higher levels of mRNA. In each case the first bar shows the value before the 6 days protocol, and the other two or three bars show the values following the treatment with the factors as indicated in the table. Data are presented as means
SEM (n ¼ 3). *p < 0.05, **p < 0.01, ***p < 0.001 (see Note 9)

4. For each factor under investigation, begin with combinations of factors set at level “3” (3 is an arbitrary designation for the initially used level, allowing decreases to levels 2 or 1 or increases to levels 4 or 5; however greater changes might be needed). This nomenclature follows that in the literature [2]. Select the value for level 3 based on values shown to be effective in the literature, whenever that information is available (see Note 4). Derive the other concentration levels (1, 2, 4, 5, etc.) from the concentration level “3” by decreasing or increasing the concentration by a factor of the square root of 10 (see Note 5). In the first round, use combinations that vary from level 3 by one step 2 or 4. 5. On days 2 and 4, add 75 μl per well of the appropriate medium plus the same factors as added on day 0. 6. On day 6, harvest the aggregates of cells and prepare total RNA by any applicable method, for example, the RNA-Bee protocol [1]. 7. Select genes whose expression will be monitored to assess the degree of differentiation of the cells in the desired lineage (see Note 6). Design primers for these genes using appropriate software. 8. Use standard qPCR techniques to measure the mRNA levels for the selected set of genes [1]. Based on an assessment of the levels of mRNA for these genes, choose a “winner” combination for this round of combinations of factors (see example in Fig. 2) (see Note 7).

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9. Repeat the process in steps 2–8 above to select another “winner” combination. Repeat the process as long as substantial increases in the expression of the selected differentiation genes are observed. 10. The process outlined above may be continued as necessary, but additionally other strategies may be employed in order to potentially improve the efficiency of arriving at an optimal combination of factors (Fig. 1) (see Note 8). These alternative strategies may restart the optimization process if no further improvement is noted using the combination of factors currently being tested (optimization is stalled). These are [1] testing the appropriate concentrations of factors to be used by assessing their effect on specific intracellular signaling pathways; [2] testing new factors for their potential effects on differentiation, before including them in the combinations being tested; [3] assessing whether assessing changes in mRNAs at times other than the standard (6 days as used here) may be useful; and [4] testing whether measuring mRNAs for other genes may be useful. For example, test genes that are in the lineage being targeted (neural) versus other lineages (e.g., mesodermal) or indicators of pluripotency. Based on the outcome of these studies, perform further rounds of testing as in steps 2–8. The following steps 11–16 outline these procedures. 11. For testing effectiveness of small-molecule factors in specific intracellular signaling pathways, use monolayer culture, e.g., in 12-well plates. Change to E8 medium containing appropriate biological factors with the addition of the small molecules under investigation. Steps 12–14 provide three examples. 12. To assess the effectiveness of dorsomorphin on BMP signaling, add recombinant BMP4 together with various concentrations of dorsomorphin [5]. Select potentially BMP-responsive target genes, such as SMAD target genes ID1 and ID3 [6, 7]. The results of this test on marmoset B8 cells are illustrated in Fig. 3. 13. To assess the effectiveness of the TGF-β/activin/nodal inhibitor SB431542, remove TGFβ-1 from the medium for culture of the cells (typically the medium contains 2 ng/ml TGFβ-1), and then add a higher level of TGF-β1 (10 ng/ml) together with the inhibitor. Use appropriate target genes, such as SERPINE1 and HES1; expression of these genes is inhibited by SB431542 [8–10] (Fig. 3). 14. The small-molecule BIO activates the Wnt pathway by inhibiting GSK3. To assess the appropriate concentration of BIO, add various concentrations in E8 medium, and assess the expression of Wnt-responsive genes such as SP5 and AXIN2 [11] (Fig. 3).

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Fig. 3 Analysis of the effects of three small-molecule factors on genes that respond to activation of three different intracellular pathways in marmoset iPS cells. In each case “0” ¼ mRNA level before treatment and “-” ¼ level following 24 h in basal medium only. DM ¼ dorsomorphin at a range of concentrations from 0 to 2 μM; B ¼ 40 ng/ml BMP4. BMP4 treatment induced a statistically significant upregulation of ID1 and ID3 mRNA; co-treatment with dorsomorphin resulted in lowered ID1, but not a statistically significant lowering of ID3. SB ¼ SB4321542 at a range of concentrations from 0 to 3 μM; T ¼ 10 ng/ml TGF-β1. Treatment with TGF-β1 increased expression of SERPINE and HES1, while SB431542 coadministration resulted in dosagedependent reduction in expression of both genes. B ¼ BIO at a range of concentrations from 0 to 2 μM. BIO increased the expression of Wnt/β-catenin targets SP5 and AXIN2 (see Note 10)

15. As indicated in Fig. 1, the cyclical “hill-climbing” process may require adjustment by adding other factors to the mix being tested. To test whether other factors may be required for optimal differentiation, perform the process in steps 2–8 above with the addition of these new factors. For example, test whether FGF2 may be replaced by other factors by omitting FGF2 and adding BIO, PD0325901, DAPT, or IWR-1 (Fig. 4). 16. As a further variation, harvest the aggregates at times other than the standard (here, we used 2 and 4 days instead of the standard 6 days) (Fig. 4).

4

Notes 1. The underlying assumptions employed in the “hill-climbing” approach have been discussed in a previous publication [1]. Versions of the original feedback control system on which this is based [2] have been used in a variety of studies [12–17]. These versions are often more complex than the approach described here because they attempt to avoid local maxima (a peak other than the global maximum). However, we make the simpler assumption that there is only a global maximum because examples in differentiation protocols have not yet shown a situation where this simple assumption has proved to be invalid. In these experiments we pick the “winning” combination of factors and concentrations by an overall assessment of the expression of a set of genes. In a future expansion of this work, this assessment

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Fig. 4 Analysis of the potential effects of including new factors (BIO, PD0325901, DAPT, IWR-1) on differentiation of marmoset iPS cells. In this case gene expression was tested after 2 and 4 days of treatment as well as 6 days; results for three representative genes, NANOG, NCAD, and ERBB3, are shown. We compared two baseline conditions (with and without FGF2; coded as 33311 and 33301, see Fig. 2) with the addition of 1 μM PD0325901, 2 μM BIO, 10 μM DAPT, or 3 μM IWR-1. Statistically significant differences are indicated as ***p < 0.001, **p < 0.01, and *p < 0.05 (see Note 11)

could potentially be done mathematically; however, at the present stage of development of this approach, we do not have sufficient information to formulate a purely mathematical method for “winner” selection. 2. The neural differentiation of pluripotent stem cells has been demonstrated under both monolayer conditions [18] and suspension culture [19]. Additionally, reports of induction of neural crest cells from human pluripotent stem cells have been reported in both monolayer and suspension conditions. In our system, one aggregate (often referred to as an embryoid body) was generated per well of a U-bottom 96-well plate, similar to those utilized in other SFEBq protocols [19]. Other cell lines may perform better under monolayer

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conditions, and therefore preliminary studies should establish which conditions are optimal for the specific cell line. 3. A significant advance in the field of neural differentiation from pluripotent cells was the introduction of the concept of FGF withdrawal coupled with dual SMAD inhibition [18]. Initially this was accomplished by the combination of noggin and SB431542, while later versions substituted chemical inhibitors such as dorsomorphin for the more expensive noggin [20, 21]. 4. In some cases, starting concentrations used may be based on an already completely defined, published protocol. In most cases this would have been defined for mouse or human pluripotent cells; while the optimal combinations and concentrations might be quite different for an NHP cell line, or other species, it is reasonable to begin with any combination shown to be effective in human or mouse pluripotent stem cells. 5. In practice the step change in concentration for most factors may be set at the square root of 10, but occasionally it may be necessary to change this; for example, concentrations may be stepped up or stepped down by a factor of 10. 6. It is important to select an appropriate set of genes for expression testing; this may be based on what is already established for differentiation in cell culture or on what is known for embryological development in the desired lineage. Preliminary testing may be needed before an appropriate set of genes is defined. 7. Any regular qPCR protocol may be used; we used the SYBR green method, but any quantitative protocol may be employed. 8. As illustrated in Fig. 1, the normal cycle (“hill-climbing” protocol) may be interspersed with alternative strategies to improve the extent of differentiation. This may be especially necessary when using NHP cells or generally any species other than human or mouse. It may be necessary to verify that an inhibitor commonly used in those species works in the NHP species under investigation or if the effective concentration range may differ substantially. Because of the complexity of many differentiation protocols and because the molecular targets of the drugs/factors used are not always known, it is not yet clear whether a simple “hill-climbing” iterative approach to optimizing differentiation will always be appropriate. More complex algorithms might be necessary. This will require enough testing to ensure that the theoretical possibility that the search becomes stalled on a local maximum or plateau rather than a global maximum is unlikely. However, if practical experience shows that this happens with some frequency, algorithms should be adjusted to avoid this. 9. The results obtained here show only modest improvements over the starting combination for this round (e.g., dorsomorphin effect on SOX1) and show that other factors are already

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optimal (e.g., FGF2). Therefore these data indicate the need for alternative strategies to be tested (Figs. 3 and 4). 10. Under monolayer culture of marmoset iPS cells, we found that dorsomorphin partially suppressed BMP4-induced transcription of a known BMP target, ID1, at 2 μM, but failed to significantly suppress ID3. These results suggest that dorsomorphin produces an incomplete inhibitory effect on BMP signaling in these marmoset iPS cells, similar to that reported for human cells [22, 23]. These data for SB431542 with marmoset iPS cells are in line with SB431542 potency reported for human cells (0.5–1 μM; ref. 23). Our data suggest that SB431542 has activin/nodal/TGF-β inhibition potency in marmoset iPS cells similar to that reported for human cells. BIO stimulated target genes at concentrations similar to that reported in other systems. 11. The figure shows the effects of introducing four new smallmolecule factors on the neuroectodermal marker NCAD, the neural crest marker ERBB3, and the pluripotency marker NANOG. The starting combination was that coded as 33311 or 33301, see table in Fig. 2, depending if FGF2 was included or not. Data were also collected for the neuroectodermal markers SOX1, PAX6, and MSI1; for the floor plate marker FOXA2; for the neural crest markers SOX10 and TFAP2A; for the endodermal marker SOX17; for the mesodermal markers BRACHYURY and TBX6; for the mesoderm/neural crest markers SNAI1, SNAI2, and SOX9; and for the ectodermal marker GBX2. The combination of BIO and PD0325901, with or without DAPT, significantly improved differentiation over the baseline conditions (with or without FGF2). PD0325901 alone, or with IWR-1, did not increase the differentiation level, thus indicating that for future rounds of combinations of factors, the role of BIO would be worth exploring. References 1. Farnsworth SL, Qiu Z, Mishra A, Hornsby PJ (2013) Directed neural differentiation of induced pluripotent stem cells from non-human primates. Exp Biol Med 238:276–284 2. Tsutsui H, Valamehr B, Hindoyan A, Qiao R, Ding X, Guo S, Witte ON, Liu X, Ho CM, Wu H (2011) An optimized small molecule inhibitor cocktail supports long-term maintenance of human embryonic stem cells. Nat Commun 2:167 3. Wu Y, Zhang Y, Mishra A, Tardif SD, Hornsby PJ (2010) Generation of induced pluripotent

stem cells from newborn marmoset skin fibroblasts. Stem Cell Res 4:180–188 4. Mishra A, Qiu Z, Farnsworth SL, Hemmi JJ, Li M, Pickering AV, Hornsby PJ (2016) Induced pluripotent stem cells from nonhuman primates. Methods Mol Biol 1357:183–193 5. Hong CC, Yu PB (2009) Applications of small molecule BMP inhibitors in physiology and disease. Cytokine Growth Factor Rev 20:409–418 6. Hollnagel A, Oehlmann V, Heymer J, Ruther U, Nordheim A (1999) Id genes are direct targets of bone morphogenetic protein

Optimization of Differentiation of Nonhuman Primate Pluripotent Cells. . . induction in embryonic stem cells. J Biol Chem 274:19838–19845 7. Ying QL, Nichols J, Chambers I, Smith A (2003) BMP induction of id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115:281–292 8. Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS (2002) SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 62:65–74 9. Farberov S, Meidan R (2016) Thrombospondin-1 affects bovine luteal function via transforming growth factor-Beta1dependent and independent actions. Biol Reprod 94:25 10. Gudey SK, Sundar R, Heldin CH, Bergh A, Landstrom M (2017) Pro-invasive properties of Snail1 are regulated by sumoylation in response to TGFbeta stimulation in cancer. Oncotarget 8:97703–97726 11. De Jaime-Soguero A, Aulicino F, Ertaylan G, Griego A, Cerrato A, Tallam A, Del Sol A, Cosma MP, Lluis F (2017) Wnt/Tcf1 pathway restricts embryonic stem cell cycle through activation of the Ink4/Arf locus. PLoS Genet 13:e1006682 12. Kang Y, Hodges A, Ong E, Roberts W, Piermarocchi C, Paternostro G (2014) Identification of drug combinations containing imatinib for treatment of BCR-ABL+ leukemias. PLoS One 9:e102221 13. Ding X, Matsuo K, Xu L, Yang J, Zheng L (2015) Optimized combinations of bortezomib, camptothecin, and doxorubicin show increased efficacy and reduced toxicity in treating oral cancer. Anti-Cancer Drugs 26:547–554 14. Weiss A, Ding X, van Beijnum JR, Wong I, Wong TJ, Berndsen RH, Dormond O, Dallinga M, Shen L, Schlingemann RO, Pili R, Ho CM, Dyson PJ, van den Bergh H, Griffioen AW, Nowak-Sliwinska P (2015) Rapid optimization of drug combinations for the optimal angiostatic treatment of cancer. Angiogenesis 18:233–244 15. Weiss A, Berndsen RH, Ding X, Ho CM, Dyson PJ, van den Bergh H, Griffioen AW,

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Nowak-Sliwinska P (2015) A streamlined search technology for identification of synergistic drug combinations. Sci Rep 5:14508 16. Liu Q, Zhang C, Ding X, Deng H, Zhang D, Cui W, Xu H, Wang Y, Xu W, Lv L, Zhang H, He Y, Wu Q, Szyf M, Ho CM, Zhu J (2015) Preclinical optimization of a broad-spectrum anti-bladder cancer tri-drug regimen via the feedback system control (FSC) platform. Sci Rep 5:11464 17. Ding X, Njus Z, Kong T, Su W, Ho CM, Pandey S (2017) Effective drug combination for Caenorhabditis elegans nematodes discovered by output-driven feedback system control technique. Sci Adv 3:eaao1254 18. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275–280 19. Eiraku M, Watanabe K, Matsuo-Takasaki M, Kawada M, Yonemura S, Matsumura M, Wataya T, Nishiyama A, Muguruma K, Sasai Y (2008) Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3:519–532 20. Zhou J, Su P, Li D, Tsang S, Duan E, Wang F (2010) High-efficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor beta superfamily receptors. Stem Cells 28:1741–1750 21. Morizane A, Doi D, Kikuchi T, Nishimura K, Takahashi J (2011) Small-molecule inhibitors of bone morphogenic protein and activin/ nodal signals promote highly efficient neural induction from human pluripotent stem cells. J Neurosci Res 89:117–126 22. Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng SA, Lin HY, Bloch KD, Peterson RT (2008) Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat Chem Biol 4:33–41 23. Vogt J, Traynor R, Sapkota GP (2011) The specificities of small molecule inhibitors of the TGF and BMP pathways. Cell Signal 23:1831–1842

Chapter 15 Isolation and Differentiation of Self-Renewable Neural Stem Cells from Marmoset-Induced Pluripotent Stem Cells Hyenjong Hong, Gourav Roy-Choudhury, Jeffrey Kim, and Marcel M. Daadi Abstract Neural stem cells (NSCs) are multipotent and self-renewing precursor cells that give rise to all cell types of the central nervous system (CNS). They can be used for modeling CNS in vitro, for developmental studies and for cell replacement therapies. NSCs can be derived from pluripotent stem cells through differentiation using specific growth factors. Nonhuman primates (NHP) are critical preclinical models for translational research. Induced pluripotent stem cells (iPSCs) can be generated from NHP for the purposes of allogenic or autologous cell replacement studies. Here, we describe the derivation of NSCs from NHP iPSCs. Key words Neural stem cells, Nonhuman primate, Induced pluripotent stem cells, Marmosets

1

Introduction Animal cell lines and models have been used to provide significant advances in our understanding of human diseases, development, and evolution. However, there are intrinsic limitations to using smaller animal models as they are not completely representative of human biology. This limitation is mitigated with the use of nonhuman primates (NHP). NHP share similar physiology to that of humans and are an important and necessary intermediary model between, for instance, humans and rodents. In the context of cell replacement therapy, having an animal model with similar physiology, size, and complexity of the human immune system is relevant in preclinical studies. Understanding the subtle intricacies between the host immune system and graft is important prior to initiation of any clinical studies using pluripotent stem-based cell therapies. IPSCs derived from NHP offer the advantage of testing novel therapeutic approaches, such as autologous cell therapy. However, reliable methods for isolating and differentiating specific cell types from iPSCs are needed to understand the similarities and differences

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between nonhuman and human primates. In this chapter, we describe a method for the derivation of NSCs from NHP iPSCs. These NHP NSCs are able to grow in suspension culture and display a similar morphology to that of NSCs derived from human iPSCs.

2 2.1

Materials Equipment

1. Cell culture incubator (NuAire, Plymouth, MN, USA). 2. Phase contrast microscope (Zeiss, Oberkochen, Germany). 3. Centrifuge (Eppendorf, Hamburg, Germany). 4. T25 tissue culture flask (Corning, Oneonta, NY, USA). 5. T75 tissue culture flask (Corning, Oneonta, NY, USA). 6. Cell lifter (Fisher Scientific, Pittsburgh, PA, USA). 7. Glass pipettes (Fisher Scientific, Pittsburgh, PA, USA). 8. Centrifuge tubes (15 mL and 50 mL) (Corning, Oneonta, NY, USA). 9. Syringe filters (Corning, Oneonta, NY, USA). 10. Syringes (10, 20, and 60 mL) (BD, Franklin Lakes, NJ, USA). 11. Pipettes (2–25 mL) (Fisher Scientific, Pittsburgh, PA, USA). 12. Pipette aids (10–1000 μL) (Eppendorf, Hamburg, Germany). 13. Pipette tips (10–1000 μL) (Accuflow, E&K Scientific, Santa Clara, CA, USA). 14. Water bath (Fisher Scientific, Pittsburgh, PA, USA). 15. Hemocytometer (Hausser Scientific, Horsham, PA, USA) or automated cell counter (Countess, Invitrogen, Carlsbad, CA, USA).

2.2

Reagents

1. NN1 media (NeoNeuron, San Antonio, TX, USA). 2. Basic fibroblast growth factor (Stemgent, Cambridge, MA, USA). 3. Epidermal growth factor (EGF, EMD Millipore, Burlington, MA, USA). 4. Retinoic acid (Sigma-Aldrich, St. Louis, MO, USA). 5. Fetal bovine serum (FBS, GE, Logan, UT, USA). 6. Poly-L-ornithine hydrobromide (Sigma-Aldrich, St. Louis, MO, USA). 7. Accutase (Gibco, Life Technologies, NY, USA). 8. Trypsin neutralizer (Gibco, Life Technologies, NY, USA). 9. Tris base (Fisher Bioreagents, Pittsburgh, PA, USA). 10. Phosphate-buffered saline (PBS, Gibco, Life Technologies, NY, USA). 11. Double-distilled water (Gibco, Life Technologies, NY, USA).

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3.1 Preparation of Reagents and Media

Prepare all the reagents under sterile conditions in a horizontal laminar flow hood 1. Preparation of 10 mM Tris (25 mL): Dissolve 30.35 mg of Tris base (F.Wt: 121.4) in 15 mL of double-distilled water and adjust the pH to 7.6. Increase the volume to 25 mL and filter sterilize. Store at 4  C (see Note 1). 2. Preparation of basic fibroblast growth factor stock solution: Briefly centrifuge the tube, and reconstitute the bFGF (50 μg) in 2.5 mL of 10 mM Tris solution (pH 7.6) to prepare a 20 μg/ mL stock solution. Aliquot and store at 20  C. 3. Preparation of epidermal growth factor stock solution: Briefly centrifuge the tube, and reconstitute the EGF (500 μg) in 5 mL of double-distilled water to prepare a stock solution of 100 μg/mL. Aliquot and store at 20  C. 4. Preparation of retinoic acid (RA) stock solution: Dissolve 3 mg of RA (F.Wt: 300.44) in 1 mL of distilled water to prepare 10 mM stock solution. Aliquot and store at 20  C. 5. Poly-L-ornithine hydrobromide (PLO): Dissolve poly-L-ornithine hydrobromide (PLO) in D.D water under sterile conditions to prepare a 10 stock solution of 0.16 mg/mL. Aliquot and store at 20  C. 6. Preparation of neural stem cell (NSC) media (50 mL): To 50 mL of NN1 media (NeoNeuron), add 50 μL of bFGF (20 μg/mL) and 10 μL of EGF (100 μg/mL) and 10 μL of RA (10 mM) to prepare NSC media with final concentration of 2 μM RA, 20 ng/mL of bFGF and EGF. Filter sterilize and store at 4  C. 7. Preparation of differentiation media (50 mL): To 50 mL of NN1 media, add 500 μL of FBS (final concentration 1%) and 10 μL of RA (10 mM). Filter sterilize and store at 4  C (see Note 2).

3.2 Isolation of NSCs from iPSCs

1. Culture and expand marmoset iPSCs (on feeder or feeder-free) in a 6-well plate until they are 80–90% confluent. 2. On the day of the experiment, pre-warm the NSC media in a 37  C water bath. 3. Nonenzymatically detach the iPSC colonies using a cell lifter. Take care not to dissociate colonies into single cells. 4. Carefully collect the colonies from the 6-well plate using a 5 mL pipette, and transfer them to a 15 mL centrifuge tube. Do not triturate the cells. 5. Centrifuge at 1000 rpm (200  g) for 5 min.

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6. Using vacuum-aided suction, carefully remove the media without disturbing the pellet using a glass pipette. 7. Resuspend the pellet in 5 mL of NSC media. 8. Centrifuge at 1000 rpm (200  g) for 5 min. 9. Resuspend in 8 mL of NSC media and plate in a T-25 flask. 10. Three days after plating or when the media turns yellow, replace old media with fresh NSC media (NN1 + 20 ng/mL bFGF + 2 ng/mL EGF + 2 μM RA) (see Note 2). 11. To change media, gently decant the media from culture flask into a 50 mL centrifuge tube. 12. Centrifuge the cells at 1000 rpm (200  g) for 5 min. 13. Carefully remove the supernatant by using vacuum suction without disturbing the pellet. 14. Resuspend the pellet with fresh NSC media and replate them in a T-25 flask. 15. Change the media every 3–4 days or as required. 16. By the end of 2 weeks, the NSCs can be seen to form neurospheres. 3.3 Coating Culture Plates with PLO

1. Under sterile conditions, prepare working solution of PLO (16 μg/mL) by diluting 1 mL of 10 stock solution (0.16 mg/mL) in 9 mL of D.D water. 2. Add PLO working solution to each well of the desired plate type (6–96 well) to cover the entire surface. We typically use the following volumes for each well plate type: Plate type

Volume (μL)

96

25–30

48

80

24

250

12

500

6

1000

3. Incubate the plate in the cell culture incubator for a minimum of 2 h. 4. After incubation, wash the plate twice with sterile PBS and plate the cells. The plates can be prepared a day in advance and stored 4  C under sterile culture conditions. 3.4 Differentiation of iPSC-Derived NSCs

Pre-warm the differentiation media in a 37  C water bath (Fig. 1). 1. Collect the floating neurospheres from the T-25 flask (see Fig. 1) into a 15 mL tube, and centrifuge at 1000 rpm (200  g) for 5 min.

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Fig. 1 Differentiation of NSCs isolated from marmoset iPSCs. (a) Representative phase-contrast image of neurospheres isolated from marmoset iPSCs. NSCs isolated from marmoset iPSCs with or without retinoic acid (RA) were differentiated for 7 days in NN1 media containing 1% FBS. Immunofluorescence staining results showed that NSCs isolated with (b) control media (without RA in NSC media) contained less number of cells positive for neuronal marker β-III-tubulin (TuJ1) compared to (c) those isolated with RA-containing NSC media

2. Carefully remove the supernatant by using vacuum suction without disturbing the pellet. 3. Gently resuspend the pellet in fresh 10 mL of fresh NN1 media and centrifuge again at 1000 rpm (200  g) for 5 min. 4. Resuspend the neurospheres (NN1 + 1% FBS).

in

differentiation

media

5. Plate the neurospheres in PLO-coated wells. We typically use the following volumes for each well plate type: Plate type

Volume (μL)

96

100

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250

24

500

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1000

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6. Day 0: The day of plating the neurospheres with differentiation media. 7. Change media every 24 h with fresh differentiation media until the completion of the experiment or transplantation.

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Notes 1. Prepare all the solutions and reagents in double-distilled water or ultrapure water (18 MΩ cm at 25  C). 2. Calculate total amount of differentiation media required for the experiment and prepare the media in advance. This will help to maintain constant concentration of all the factors during differentiation.

Acknowledgments The authors thank members of the Daadi laboratory for helpful support and suggestions. This work was supported by the Worth Family Fund, the Perry & Ruby Stevens Charitable Foundation and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation, the NIH primate center base grant (Office of Research Infrastructure Programs/OD P51 OD011133), the National Center for Advancing Translational Sciences, and the National Institutes of Health, through Grant UL1 TR001120. Disclosures: Dr. Marcel M. Daadi is founder of the biotech company NeoNeuron.

Chapter 16 Lentiviral Infection of Mouse Bone Marrow Cells for Hematopoietic Stem Cell Transplantation Cang Chen, Michael J. Guderyon, Guo Ge, Robert A. Clark, and Senlin Li Abstract Lentiviral vectors are increasingly used as efficient gene transfer tools in the experimental and clinical gene therapy treatment of acquired and inherited genetic diseases. Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal, as well as multi-lineage differentiation and maintenance of the lymphohematopoietic system throughout life. As such, HSC transplantation (HSCT) has proven to be a powerful therapeutic modality for the treatment of both malignant and nonmalignant disorders. Transduction of lentiviral vectors into HSCs may offer long-term stable expression of a therapeutic gene in both preclinical and clinical settings. The purpose of this chapter is to describe an optimized procedure for lentiviral transduction of mouse HSCs followed by HSCT. Key words Lentiviral vector, Co-transfection, Viral transduction, Bone marrow cells, Hematopoietic stem cell transplantation

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Introduction Hematopoietic stem cell transplantation (HSCT), the transplantation of multipotent hematopoietic stem cells (HSCs), is a proven modality of treatment for a variety of human diseases in the clinic [1, 2]. The transplanted/engrafted HSCs repopulate the recipient’s entire lymphohematopoietic system. HSCT, commonly referred to as bone marrow transplantation (BMT) because donor HSCs are collected from the bone marrow, especially in the past, and secondly since donor cells will be engrafted/hosted in the bone marrow. Today HSCs are, more often than not, isolated from the blood, instead of the bone marrow [2]. BMT is also used to investigate genetic, molecular, cellular, and circuital mechanisms of pathogenesis and to develop new therapies in animal systems, particularly mouse models [3]. Lentiviral hematopoietic stem cell gene therapy has been tested successfully with clinical efficacy for several monogenetic disorders [4–7]. Similarly, ex vivo lentiviral transduction of enriched mouse HSCs followed by transplantation

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_16, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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has been performed widely in biomedical research, with the expectation of more frequent use as applications become standardized. This protocol, which has served our laboratory well over the past decade [8–12], will hopefully be useful to other investigators in various fields in this new era of cell and gene therapy/genome editing.

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Materials Working with lentiviral vectors must follow the Centers for Disease Control and Prevention (CDC) guidelines for “Biosafety in Microbiological and Biomedical Laboratories” and adhere to the National Institutes of Health (NIH) requirements of “Biosafety Considerations for Research with Lentiviral Vectors.” The following safety equipment must be used when working with lentiviruses, (1) certified class II biological safety cabinets, (2) sealed centrifuge rotors and tubes, and (3) vacuum lines equipped with in-line HEPA filters. The following personal protective equipment (PPE) must be worn when working with lentiviruses, double gloves, lab coats, and eye protection.

2.1 293T Cell Culture Medium

2.2 Bone Marrow Flushing Medium

GIBCO Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S 5000 U/mL). 1. Add 17.7 g powdered GIBCO Iscove’s Modified Dulbecco’s Medium (IMDM) to a 1 L glass beaker containing 950 mL of distilled water, and mix by gently stirring. 2. Add 3.024 g of sodium bicarbonate to the dissolved medium and continue stirring. 3. Bring the solution to a final volume of 1 L with distilled water and mix well. 4. Filter the medium through a 0.22 μm pore Corning® Vacuum Filter System. 5. Add 5 mL of heparin sodium solution (McKesson Medical Surgical, Atlanta, GA) to the filtered IMDM medium to make a 0.5% final concentration of heparin.

2.3 Other Media, Chemicals, and Reagents

1. GIBCO Opti-MEM™ I Reduced Serum Medium and GIBCO StemPro®-34 SFM Complete Medium (Invitrogen). 2. GIBCO Dulbecco’s Invitrogen).

phosphate-buffered

saline

(DPBS,

3. FuGENE® 6 Transfection Reagent (Promega, Madison, WI). 4. Lympholyte®-M Cell Separation Media (CEDARLANE®, Burlington, NC).

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5. RetroNectin® (Takara Bio, Mountain View, CA). 6. 5-Fluorouracil (5-FU) and protamine sulfate (Sigma, St. Louis, MO). 7. Murine interleukin-3, human interleukin-6, murine interleukin-1α, and murine stem cell factor (PeproTech, Rocky Hill, NJ). 2.4 Sterilized Surgical Instruments

1. Forceps. 2. Scissors. 3. Gauze.

2.5 Consumable Supplies

1. 1 mL and 10 mL syringes. 2. 26 G 1/2 in. and 30 G 1/2 in. needles. 3. T-150 cell culture flasks. 4. 0.22 μm pore Corning® Vacuum Filter System. 5. 0.45 μm pore Corning® Vacuum Filter System. 6. Nalgene 50 mL Centrifuge tube.

Oak

Ridge

High-Speed

PPCO

7. 3.5 mL Quick-Seal Polypropylene Ultracentrifuge Tube. 8. 70 μm and 100 μm cell strainers.

3

Methods Figure 1 illustrates the workflow.

3.1 293T Cell Culture for Transfection

1. Seed 5.0  106 293T cells per T-150 flask in 20 mL of 293T cell culture medium, and incubate cells at 37  C in a 5% CO2 incubator. 2. Expand cell cultures by subculturing until achieving the number of T-150 flask needed.

3.2 Lentiviral Vector Co-transfection

1. 293T cell cultures should be 50–70% confluent on the day of lentiviral vector co-transfection. 2. Take out the transfer plasmid DNA from 20  C storage, three lentiviral packaging plasmids (pMDLg/pRRE, pRSV/Rev., and pMD2G), FuGENE® 6 Transfection Reagent, OptiMEM™ I Reduced Serum Medium, and Nuclease-Free Water. 3. Prepare the transfection complex in a ratio of 10:6:1.5:2.5 of transfer vector, pMDLg/pRRE, pRSV/Rev., and pMD2G and in a 3:1 ratio of FuGENE® 6 Transfection Reagent (μL) to total plasmid DNA (μg).

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Fig. 1 Flowchart of lentiviral transduction and transplantation of mouse hematopoietic stem cells

4. Dilute FuGENE® 6 Reagent with Opti-MEM™ I Reduced Serum Medium by pipetting FuGENE® 6 Reagent directly into the medium without touching the wall of the tube (see Note 1), vortex for 1 s, and incubate at room temperature for 5 min. 5. Add the plasmid DNA mixture diluted with nuclease-free water into the diluted FuGENE® 6 Reagent, vortex for 1 s, and incubate the transfection complex for 15 min at room temperature. 6. Remove the T-150 flask of 293T cell cultures from the tissue incubator, add the transfection complex to the cells in a dropwise manner (see Note 2), and gently rock the flasks in a horizontal direction several times to stir the solution before returning the cell cultures to the incubator. 3.3 Lentiviral Particle Production and Concentration

1. Twenty four hours post-lentiviral vector co-transfection, collect 20 mL of 293T cell culture medium containing lentiviral particles from each T-150 flask into 50 mL centrifuge tubes, add 20 mL of fresh 293T cell culture medium into each of the flasks, and continue incubation of the transfected 293T cells for up to 48 h (see Notes 3 and 4). 2. Centrifuge the collected 293T cell culture medium in 50 mL tubes at 433  g, 4  C for 15 min.

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3. Filter the supernatant containing lentiviral particles through a 0.45 μm pore size filter unit and store at 4  C. 4. Repeat steps 1–3 at 48 h post-lentiviral vector co-transfection, and pool the filtered supernatant with the stored supernatant (step 3). 5. Centrifuge the pooled supernatant/lentiviral particles in Nalgene High-Speed Centrifuge tubes via high-speed centrifugation at 56,000  g, 4  C for 2 h. 6. Resuspend the viral pellet with 3.5 mL of StemPro®-34 SFM medium, and transfer the viral suspension into Quick-Seal Polypropylene Ultracentrifuge Tube. 7. Centrifuge the viral suspension via ultracentrifugation at 72,000  g, 4  C for 1.5 h. 8. Discard the supernatant, and resuspend the viral pellet in a small volume (0.8–1.0 mL) of StemPro®-34 SFM medium containing 1% L-glutamine and 1% P/S. 9. Transfer the concentrated viral suspension into a 1.5 mL microcentrifuge tube and store at 80  C until use. 3.4 Coating Cell Culture Plate with RetroNectin®

1. Prepare a solution of 50 μg/mL RetroNectin in DPBS and filter it through 0.22 μm sterile syringe filter. 2. Make 2% BSA solution in DPBS and filter it through 0.22 μm sterile syringe filter. 3. Deliver 0.5 mL of 50 μg/mL RetroNectin solution into 1 well of a FALCON® 24-well non-tissue culture-treated plate, and keep the plate in a tissue culture hood at room temperature for 2 h. 4. Remove the RetroNectin solution and add 0.5 mL of BSA solution to the well for 30 min. 5. Remove the BSA solution from the well and keep the RetroNectin-coated plate at 4  C until use.

3.5 Preparation of Donor Bone Marrow Cells for Lentiviral Infection

1. Four days prior to bone marrow-derived HSC transplantation (HSCT), administer 150 mg/kg of 5-FU via tail vein injections into donor mice for enrichment of hematopoietic stem cells (HSCs) [8] (see Note 5). 2. One day prior to HSCT, donor mice are euthanatized by cervical dislocation (preferably). Isolate femurs, tibias, humeri, and hip bones, and place them in petri dishes containing IMDM. 3. Remove adherent muscle tissue with scissors and gauze. 4. Carefully cut the bones at each side with the scissors, and flush the bone marrow from each side into a 50 mL conical tube with bone marrow flushing medium using a 10 mL syringe and 26G 1/2 in. needle.

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5. Filter bone marrow cell (BMC) suspension through a sterile 100 μm cell strainer into a fresh 50 mL conical tube. 6. Centrifuge the tube at 433  g, 4  C for 5 min and discard the supernatant. 7. Resuspend the BMC pellet in a proper volume of IMDM. Slowly load 5 mL cell suspension onto a 15 mL conical tube already containing 5 mL of Lympholyte®-M Cell Separation Media (see Note 6). 8. Centrifuge the tube at 1400  g without brake for 20 min at room temperature (see Note 7). 9. After centrifugation, carefully remove and discard top medium, collect the middle cell layer from the interface using a Pasteur pipette, and transfer to a fresh 50 mL conical tube containing 20 mL of IMDM. 10. Centrifuge the tube at 800  g, 4  C for 10 min. Resuspend cells in IMDM and centrifuge the tube again at 433  g, 4  C for 5 min twice (see Note 8). 11. Resuspend cells in StemPro®-34 SFM medium supplemented with 2 mmol/L L-glutamine, 6 ng/mL of murine interleukin3, 10 ng/mL of human interleukin-6, 10 ng/mL of murine interleukin-1α, and 100 ng/mL of murine stem cell factor, and culture the enriched bone marrow-derived HSCs at concentration of 1 x 107 cells/10 mL per petri dish in a 5% CO2 incubator at 37  C overnight. 3.6 Lentiviral Infection

1. Take out the RetroNectin-coated plate and thaw the frozen concentrated lentiviruses. Add cytokines and protamine to the lentiviral suspension at final concentrations of 6 ng/mL murine interleukin-3, 10 ng/mL human interleukin-6, 10 ng/mL murine interleukin-1α, 100 ng/mL murine stem cell factor, and 8 μg/mL protamine. 2. Collect overnight-cultured donor bone marrow-derived HSCs in a 50 mL conical tube, and centrifuge the tube at 433  g, 4  C for 5 min. 3. Resuspend the donor cells with 0.8 mL of lentiviral suspension (see Note 9), transfer the cell suspension into the well coated with RetroNectin, and incubate the cells with the lentiviruses in a 5% CO2 incubator at 37  C for 6 h.

3.7 Preparation of Recipient Mice for HSC Transplantation

1. Three days prior to HSCT, administer acidic drinking water (pH 3.0) containing 50 mg Neomycin Sulfate and 5 mg polymyxin B sulfate to recipient mice. 2. Six hours prior to HSCT, the recipient mice are irradiated at 950 cGy (whole body or partial) from a gamma irradiation source.

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1. After 6-h lentiviral infection, collect donor cells from the infection culture well, and filter the cell suspension through a sterile 70 μm cell strainer into a fresh 50 mL conical tube along with StemPro®-34 SFM medium (see Note 10). 2. Centrifuge the tube at 433  g, 4  C for 5 min, resuspend the cells in a proper volume of StemPro®-34 SFM medium, and count the cells harvested. 3. Centrifuge the tube at 433  g, 4  C for 5 min, and resuspend the cells in a calculated volume of DPBS containing 2% FBS to a final concentration of 3  106 cells/0.2 mL. 4. Inject 3  106 cells/0.2 mL into each irradiated recipient mouse via tail vein injections (see Note 11).

3.9 Care for Recipient Mice Post-transplantation

1. After transplantation, the recipient mice should be maintained on acidic drinking water containing 50 mg neomycin sulfate and 5 mg polymyxin B sulfate for 3 weeks. 2. Change cages with fresh food every other day for recipient mice.

3.10 Assessment of HSCT Efficacy

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The efficacy of transplantation/engraftment of lentiviral vectortransduced HSCs can be determined via flow cytometry for the percentage of donor cells stained with specific antibodies in peripheral blood at 1 and 4 months after HSCT.

Notes 1. Do not aliquot FuGENE® 6 reagent from the original glass vials because chemical residues in plastic vials can significantly decrease the biological activity of the reagent. Do not use siliconized pipette tips or tubes for handling of FuGENE® 6 Reagent. Avoid touching the wall of plastic tube while delivering FuGENE® 6 Reagent into Opti-MEM™ I medium. 2. Hold the 293T cell culture flask side-straight up at a 30-degree angle to add the transfection complex into the culture medium in a dropwise manner using a 2 mL serological pipette without scratching the cell layer on the bottom of the flask. 3. Gently add fresh medium from the side of culture flask and avoid the cell layer coming off at the time of first virus collection. 4. The transfection efficiency can be estimated at 24 h posttransfection. We always set up a lentiviral GFP transfer vector co-transfection in parallel to facilitate evaluation of the transfection efficiency (Fig. 2).

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Fig. 2 293T cells at 24 h after lentiviral GFP vector co-transfection with packaging plasmids. (a) Differential interference contrast (DIC) image. (b) GFP fluorescence image showing 80–90% transfection efficiency achieved

5. It is very difficult to dissolve 5-FU powder in physiological saline. We add the 5-FU powder into a 15 mL conical tube containing physiological saline, place the tube in a 55  C water bath, vigorously vortex tube from time to time until 5-FU is dissolved completely, and filter the solution through a 0.22 μm sterile syringe filter. 6. Hold the 15 mL tube at a 30-degree angle, and carefully load the cell suspension against the wall of the tube onto the top of Lympholyte®-M Cell Media to form a distinct interface. 7. Centrifugation at 4  C may result in cell clumping or poor recovery. 8. After isolation of the cell layer, we usually wash 2 or 3 times with IMDM to remove any remaining Lympholyte®-M solution from harvested cells. 9. About 400  106 cells are usually obtained here if 10 C57BL/ 6J mice are used as donor. 10. After lentiviral infection, the cell suspension appears viscous and yellowish. Typically, two or three StemPro®-34 SFM medium washes through the 70 μm cell strainer are needed to remove viral particles and cell aggregates, and obtain a uniform single-cell suspension. 11. Mouse tail vein injection is a critical step in the HSCT procedure. Place the mouse in a restrainer. Draw the cell suspension with a 1 mL syringe and 30 G 1/2 in. needle. Ensure air bubbles are removed prior to injection. Swab the mouse tail with an alcohol pad to increase the visibility of the tail vein. Slightly rotate the tail to visualize the lateral vein. Insert the needle into the vein with bevel facing up and keep the needle

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and syringe parallel to the tail. A successful tail vein injection should feel smooth and without resistance when pressing the syringe plunger.

Acknowledgment The establishment of this method was supported by a Merit Review grant (2I01BX000737) from the Department of Veterans Affairs Biomedical Laboratory Research & Development Program and by the William and Ella Owens Medical Research Foundation. References 1. Copelan EA (2006) Hematopoietic stem-cell transplantation. N Engl J Med 354:1813–1826 2. Gratwohl A, Baldomero H, Aljurf M (2010) Hematopoietic stem cell transplantation, a global perspective. JAMA 303:1617–1624 3. Linton MF, Atkinson JB, Fazio S (1995) Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation. Science 267:1034–1037 4. Aiuti A, Biasco L, Scaramuzza S, Ferrua F, Cicalese MP, Baricordi C, Dionisio F, Calabria A, Giannelli S, Castiello MC (2013) Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science 341:1233151 5. De Ravin SS, Wu X, Moir S, Anaya-Ο’Brien S, Kwatemaa N, Littel P, Theobald N, Choi U, Su L, Marquesen M (2016) Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency. Sci Transl Med 8:335ra357 6. Biffi A, Montini E, Lorioli L, Cesani M, Fumagalli F, Plati T, Baldoli C, Martino S, Calabria A, Canale S (2013) Lentiviral hematopoietic stem cell gene therapy benefits metachromatic Leukodystrophy. Science 341:1233158 7. Sessa M, Lorioli L, Fumagalli F, Acquati S, Redaelli D, Baldoli C, Canale S, Lopez ID, Morena F, Calabria A (2016) Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy, an ad-hoc

analysis of a non-randomised, open-label, phase 1/2 trial. Lancet 388:476–487 8. He W, Qiang M, Ma W, Valente AJ, Quinones MP, Wang W, Reddick RL, Xiao Q, Ahuja SS, Clark RA (2006) Development of a synthetic promoter for macrophage gene therapy. Hum Gene Ther 17:949–959 9. Biju K, Zhou Q, Li G, Imam SZ, Roberts JL, Morgan WW, Clark RA, Li S (2010) Macrophage-mediated GDNF delivery protects against dopaminergic neurodegeneration, a therapeutic strategy for Parkinson’s disease. Mol Ther 18:1536–1544 10. Biju KC, Santacruz RA, Chen C, Zhou Q, Yao J, Rohrabaugh SL, Clark RA, Roberts JL, Phillips KA, Imam SZ (2013) Bone marrowderived microglia-based neurturin delivery protects against dopaminergic neurodegeneration in a mouse model of Parkinson’s disease. Neurosci Lett 535:24–29 11. Chen C, Li X, Ge G, Liu J, Biju KC, Laing SD, Qian Y, Ballard C, He Z, Masliah E (2018) GDNF-expressing macrophages mitigate loss of dopamine neurons and improve Parkinsonian symptoms in MitoPark mice. Sci Rep 8:5460 12. Pawliuk R, Westerman KA, Fabry ME, Payen E, Tighe R, Bouhassira EE, Acharya SA, Ellis J, London IM, Eaves CJ (2001) Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 294:2368–2371

Chapter 17 Central and Peripheral Secondary Cell Death Processes after Transient Global Ischemia in Nonhuman Primate Cerebellum and Heart Jea-Young Lee, Roger Lin, Hung Nguyen, Eleonora Russo, M. Grant Liska, Trenton Lippert, Yuji Kaneko, and Cesar V. Borlongan Abstract Cerebral ischemia and its pathological sequelae are responsible for severe neurological deficits generally attributed to the neural death within the infarcted tissue and adjacent regions. Distal brain regions, and even peripheral organs, may be subject to more subtle consequences of the primary ischemic event which can initiate parallel disease processes and promote comorbid symptomology. In order to characterize the susceptibility of cerebellar brain regions and the heart to transient global ischemia (TGI) in nonhuman primates (NHP), brain and heart tissues were harvested 6 months post-TGI injury. Immunostaining analysis with unbiased stereology revealed significant cell death in lobule III and IX of the TGI cerebellum when compared to sham cerebellum, coinciding with an increase in inflammatory and apoptotic markers. Cardiac tissue analysis showed similar increases in inflammatory and apoptotic cells within TGI hearts. A progressive inflammatory response and cell death within the cerebellum and heart of chronic TGI NHPs indicate secondary injury processes manifesting both centrally and peripherally. This understanding of distal disease processes of cerebral ischemia underscores the importance of the chronic aberrant inflammatory response and emphasizes the needs for therapeutic options tailored to target these pathways. Here, we discuss the protocols for characterizing the histopathological effects of transient global ischemia in nonhuman primate cerebellum and heart, with an emphasis on the inflammatory and apoptotic cell death processes. Key words Secondary injury, Neurodegeneration, Cell death, Purkinje cells, Inflammation

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Introduction The global ischemia resulting from circulatory arrest is understood to instigate pathological changes within the human brain [1], processes which have been studied using both nonsurgical [2, 3] and surgical methods [4]. Advances in animal modeling have allowed scientists to safely and effectively reproduce transient global ischemia (TGI) via noninvasive surgical techniques [5]. Investigations have revealed that global ischemia is directly

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_17, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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associated with focal neuronal damage in susceptible brain regions such as the hippocampus and neocortex [5, 6]. Potential pathological links may exist between this neuronal death caused by cerebral ischemia and cardiac myocyte vulnerability [7–9]. Supporting this, in vivo murine models and in vitro studies have found an upregulation of cell death cues in cardiac tissue following cerebral ischemia [7, 10]. This knowledge is likely implicated in the increased incidence of cardiac-related deaths observed in humans within 3 months of cerebral ischemia [7, 11, 12]. Our laboratory has demonstrated with rodent models that cerebral ischemia produces secondary cellular damage in peripheral organs, particularly the heart [7]. In search of a mechanistic link, in vitro experimentation revealed that oxygen-glucose deprivation (OGD) of primary rat neuronal cells caused a significant decrease in viability of co-cultured cardiomyocytes, with the results of in vivo histology supporting this notion [7]. Together, these results advance the support for a pathological link between cerebral ischemia and latent cardiovascular disease by way of indirect cell death signals emanating from the primary cerebral injury. Cerebrovascular disease and cardiovascular disease exhibit numerous common predisposing factors, such as diabetes, hyperlipidemia, hypertension, and genetic factors [13–21]. Studies have revealed an increased incidence of cardiac cell death following cerebrovascular accident, potentially a result of elevated plasma catecholamine and cardiac enzymes (including troponin and creatine phosphokinase) [11, 12]. Moreover, severe ischemic insults are associated with heightened levels of brain natriuretic peptide (BNP) [13–21], indicating the presence of molecular crosstalk between the brain and heart following cerebrovascular ischemia [19–22]. Inflammatory signals may serve similar roles as a pathological link between the brain and heart; the plasma levels of C-reactive protein (CRP), which are known to be increased during periods of inflammation, have been established as a specific risk factor connected to new cardiovascular events in recovering stroke patients [12], indicating the inflammatory system as a key mediator of cardiovascular disease following stroke. In a quest to better understand the molecular, cellular, and anatomical links between these two pathological conditions, highly translatable models of ischemic stroke such as the nonhuman primate model of cerebral ischemia may facilitate an improved understanding of the cerebrovascular events and its damaging effects on the heart. The present protocol allows for analysis of the brain and heart tissue of NHPs which have suffered chronic TGI, with an emphasis on the inflammatory mediators which may mediate the secondary cardiovascular damage. This line of investigation may further our understanding of cerebrovascular disease and cardiovascular disease as intertwined disease states which may see clinical benefit from an integrated approach. The present chapter, based on

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our previous report [23], details the protocols necessary to reveal the histopathological effects of transient global ischemia in nonhuman primate cerebellum and heart, with an emphasis on the inflammatory and apoptotic cellular alterations.

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Materials

2.1 Transient Global Ischemia Surgery

1. Anesthetic machine with 1–2% halothane. 2. Scalpel and hemostats. 3. Thermal blanket pad. 4. Ketoprofen analgesic. 5. Rectal thermometer.

2.2 Brain and Organ Harvesting, Fixation, and Sectioning

1. Cold phosphate-buffered saline (PBS). 2. Cold 4% paraformaldehyde in phosphate buffer (PB). 3. Peristaltic pump. 4. 30% sucrose. 5. OCT embedding compound. 6. Cryostat. 7. Cryoprotectant solution. 8. Glass slides.

2.3 Immunohistochemistry

1. Anti-calbindin antibody. 2. 0.1 M PBS. 3. 2% hydrogen peroxide. 4. 40% methanol. 5. 10% normal goat serum. 6. 0.1% Triton X-100. 7. Rabbit anti-calbindin (1:300; Cell Signaling). 8. 3% normal serum. 9. Biotinylated goat anti-rabbit secondary antibody (1:200; Vector Laboratories, Burlingame, CA). 10. Avidin-biotin substrate (ABC kit, Vector Laboratories, Burlingame, CA). 11. 3,30 -diaminobenzidine (DAB) without metal enhancer (Vector Laboratories). 12. Glass slides. 13. Ethanol solutions (70%, 95%, and 100%). 14. Xylenes. 15. Toluene.

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2.4 Immunofluorescent Staining

1. Saline sodium citrate (SSC). 2. 8% normal goat serum (Invitrogen). 3. 0.1 M PBS containing 0.1% Tween 20 (PBST) (Sigma). 4. Rabbit polyclonal Technologies).

anti-human

TNF-α

(1:200;

Life

5. Chicken polyclonal anti-human GFAP (1:100; Abcam). 6. 5% normal goat serum. 7. Goat anti-rabbit IgG-Alexa 594 (red) (1:1500; Invitrogen). 8. Goat anti-chicken IgG-Alexa 488 (green) (1:400; Invitrogen). 9. Hoechst 33258 (1:300; Sigma). 10. Fluoromount (Sigma). 11. Confocal microscope (Olympus). 12. 2% hydrogen peroxide. 13. 0.1% Triton X-100. 14. Anti-human CD68 (1:100; Bio-Rad). 15. HLA-DR (1:600; Dako). 16. Rabbit polyclonal Technologies).

anti-human

TNF-α

(1:200;

Life

17. Goat anti-mouse IgG-Alexa 488 (green) (1:400; Invitrogen). 18. Goat anti-rabbit IgG-Alexa 594 (red) (1:1500; Invitrogen). 19. Rabbit polyclonal anti-human caspase 3 (1:250) antibody markers. 2.5 Stereological Analysis: Cavalieri Estimator

1. Cavalieri estimator probe.

2.6 Analysis of Fluorescent Staining

1. ImageJ (National Institutes of Health).

2.7 Statistical Analysis

1. Bonferroni’s test (GraphPad).

3

2. Pearson correlation test.

Methods

3.1 Transient Global Ischemia Surgery

1. Sedate the NHPs with ketamine (10 mg/kg) IM and pre-anesthetize with atropine (0.02 mg/kg) SC. 2. Intubate the animals with appropriate sized endotracheal tube. 3. Maintain the NHPs under anesthesia through the surgical procedure with 1–2% isoflurane in nitrous oxide/oxygen (59%/40%) mixture (see Note 1).

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4. Maintain the NHPs’ body temperature at 37  0.3  C using rectal thermometer and heating blanket. 5. Monitor appropriate physiological parameters such as body temperature, pupil size, respiratory rate, blood pressure, SPO2, etc. (see Note 2). 6. Prepare the NHPs by shaving the thoracic and abdomen regions, followed by appropriate aseptic cleaning steps. 7. Expose the innominate and left subclavian arteries using thoraco-laparotomy techniques (see Note 3). 8. Clip the arteries distal to the bifurcation from the aortic arch for 20 min. 9. For sham surgery, anesthetize the animals and expose the arties but without clipping. 10. Administer analgesic compound after the surgery and as needed thereafter (see Note 1). 11. Monitor the animals closely with weight and health surveillance recording as per IACUC guidelines. 3.2 Brain and Organ Harvesting, Fixation, and Sectioning

1. Under deep anesthesia, re-anesthetize NHPs (see Note 4). 2. Perfuse NHPs through the ascending aorta with 500 mL of saline, followed by 1000 mL of 4% paraformaldehyde in phosphate buffer (PB). 3. Collect the cerebellum and the hearts and postfix in the same fixative for 24 h, followed by 30% sucrose in PB until completely sunk (see Note 5). 4. Cut the cerebellum (the vermis and the hemisphere) in series of lateral sections at a thickness of 40 μm with a cryostat, and store at 20  C in cryoprotectant solution. 5. Cut the heart in series of transverse sections from the short axis of the base (ventricles) to the apex at a thickness of 40 μm, and mount directly onto slides and store at 20  C.

3.3 Immunohistochemistry Staining for Calbindin of the Cerebellar

1. Select every sixth 40 μm lateral section of the vermis and cerebellar hemispheres and anatomically match the sections. 2. Wash six free-floating coronal sections three times in 0.1 M phosphate-buffered saline (PBS). 3. Incubate all sections in 2% hydrogen peroxide (H2O2) and 40% methanol solution for 20 min. 4. Wash three times with 0.1 M PBS for 10 min each wash. 5. Incubate in blocking solution for 1 h using 0.1 M PBS supplemented with 10% normal goat serum and 0.1% Triton X-100.

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6. Incubate overnight at 4  C with rabbit anti-calbindin (1:300; cell signaling, 2173), antibody markers in 0.1 M PBS supplemented with 3% normal serum and 0.1% Triton X-100. 7. Wash three times with 0.1 M PBS. 8. Incubate in biotinylated goat anti-rabbit secondary antibody (1:200; Vector Laboratories, Burlingame, CA) in 0.1 M PBS supplemented with normal goat serum and 0.1% Triton X-100 for 1 h. 9. Wash three times with 0.1 M PBS. 10. Incubate for 1 h in avidin-biotin substrate (ABC kit, Vector Laboratories, Burlingame, CA). 11. Wash three times with 0.1 M PBS for 10 min each wash. 12. Incubate for 1 min in 3,30 -diaminobenzidine (DAB) without metal enhancer (Vector Laboratories). 13. Wash three times with 0.1 M PBS for 10 min each wash. 14. Mount onto glass slides and dehydrate in ascending ethanol concentration (70%, 95%, and 100%) for 2 min each and 2 min in xylenes, and coverslip using toluene as mounting medium. 3.4 Immunofluorescent Staining for GFAP and TNF-α of the Cerebellar

1. Select every sixth 40 μm lateral section of the vermis and cerebellar hemispheres and anatomically match the sections. 2. Wash three times for 10 min in 0.1 M PBS. 3. Incubate with saline sodium citrate (SSC) solution at pH 6 for 40 min at 80  C for antigen retrieval. 4. Treat with blocking solution for 60 min at room temperature with 8% normal goat serum (Invitrogen, CA) in 0.1 M PBS containing 0.1% Tween 20 (PBST) (Sigma). 5. Incubate overnight at 4  C with rabbit polyclonal anti-human TNF-α (1:200; Life Technologies, PA1-26810) and chicken polyclonal anti-human GFAP (1:100; Abcam, ab4674) with 3% normal goat serum (see Note 6). 6. Wash five times for 10 min in 0.1 M PBST, and soak in 5% normal goat serum in 0.1 M PBST containing corresponding secondary antibodies, goat anti-rabbit IgG-Alexa 594 (red) (1:1500; Invitrogen), and in goat anti-chicken IgG-Alexa 488 (green) (1:400; Invitrogen) for 90 min. 7. Wash five times for 10 min in 0.1 M PBST and three times for 5 min in 0.1 M PBS. 8. Process for 1:300 Hoechst 33258 (bisBenzimideH 33258 trihydrochloride, Sigma) for 30 min. Wash with 0.1 M PBS, and coverslip with Fluoromount (Aqueous Mounting Medium; sigma F4680) (see Note 7).

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9. Examine using a confocal microscope (Olympus). Control studies include exclusion of primary antibody substitute with 5% normal goat serum in 0.1 M PBS. No immunoreactivity should be observed in these controls. 3.5 Immunofluorescent Staining for CD68, HLA-DR, and TNF-α of the Heart

1. For CD68, HLA-DR, and TNF-α staining, wash four transverse sections of the base of the heart (40 μm) six times in 0.1 M phosphate-buffered saline (PBS). 2. Incubate with saline sodium citrate (SSC) solution at pH 6 for 40 min at 80  C for antigen retrieval. 3. Soak in 2% hydrogen peroxide (H2O2) solution for 20 min, and then wash three times with 0.1 M PBS for 10 min each wash. 4. Incubate in blocking solution for 1 h using 0.1 M PBS supplemented with 5% normal goat serum (Invitrogen, CA) and 0.1% Triton X-100. 5. Incubate overnight at 4  C with mouse anti-human CD68 (1:100; Bio-Rad MCA341), HLA-DR (1:600; Dako M0746), and rabbit polyclonal anti-human TNF-α (1:200; Life Technologies, PA1-26810) antibody markers in 0.1 M PBS supplemented with 3% normal serum and 0.1% Triton X-100 (see Note 6). 6. Wash three times with 0.1 M PBS, and incubate with goat antimouse IgG-Alexa 488 (green) (1:400; Invitrogen) and goat anti-rabbit IgG-Alexa 594 (red) (1:1500; Invitrogen) in 0.1 M PBS supplemented with normal goat serum and 0.1% Triton X-100 for 1 h. 7. Wash five times for 10 min in 0.1 M PBST and three times for 5 min in 0.1 M PBS. 8. Process for 1:300 Hoechst 33258 (bisBenzimideH 33258 trihydrochloride, Sigma) for 30 min, wash with 0.1 M PBS, and coverslip with Fluoromount (Aqueous Mounting Medium; Sigma F4680) (see Note 7). 9. Examine using a confocal microscope (Olympus). Control studies include exclusion of primary antibody substituted with 5% normal goat serum in 0.1 M PBS. No immunoreactivity should be observed in these controls.

3.6 Immunofluorescent Staining for HLA-DR and Caspase 3 of the Heart

1. For HLA-DR and caspase 3 staining, wash four transverse sections of the base of the heart (40 μm) six times in 0.1 M phosphate-buffered saline (PBS). 2. Incubate with saline sodium citrate (SSC) solution at pH 6 for 40 min at 80  C for antigen retrieval. 3. Subject to 2% hydrogen peroxide (H2O2) solution for 20 min and washing three times with 0.1 M PBS for 10 min each wash.

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4. Incubate in blocking solution for 1 h using 0.1 M PBS supplement with 5% normal goat serum (Invitrogen, CA) and 0.1% Triton X-100. 5. Incubate overnight at 4  C with mouse anti-human HLA-DR (human leukocyte antigen; 1:600; Dako M0746) and rabbit polyclonal anti-human caspase 3 (1:250) antibody markers in 0.1 M PBS supplemented with 3% normal serum and 0.1% Triton X-100. 6. Wash three times with 0.1 M PBS, and incubate in goat antimouse IgG-Alexa 488 (green) (1:400; Invitrogen) and goat anti-rabbit IgG-Alexa 594 (red) (1:1500; Invitrogen) in 0.1 M PBS supplement with normal goat serum and 0.1% Triton X-100 for 1 h. 7. Wash three times with 0.1 M PBS for 10 min each wash. 8. Wash five times for 10 min in PBST and three times for 5 min in PBS, process for Hoechst 33258 (bisBenzimideH 33258 trihydrochloride, Sigma) for 30 min, wash with PBS, and coverslip with Fluoromount (Aqueous Mounting Medium; Sigma F4680) (see Note 7). 9. Examine using a confocal microscope (Olympus). Control studies include exclusion of primary antibody substituted with 5% normal goat serum in 0.1 M PBS. No immunoreactivity should be observed in these controls. 3.7 Stereological Analysis: Cavalieri Estimator

1. Perform unbiased stereology on cerebellar sections immunostained with calbindin to estimate the volume of Purkinje cells. Take sets of 1/6 section, ~6 systematically random sections, about 240 μm apart, of TGI and sham cerebellum (see Note 8). 2. Examine calbindin-positive cells via the Cavalieri estimator probe of the unbiased stereological cell technique revealing the volume of calbindin-positive neurons in the cerebellum. Optimize all samplings to count at least 300 cells per cerebellar hemisphere with error coefficients less than 0.07. 3. Use a point grid spaced equally both across and down directions of Cavalieri estimator. 4. Use 100 μm as a grid space to cover the different gray and white matter regions each one representing the region of interest (ROI).

3.8 Analysis of Fluorescent Staining

1. Take approximately four to six confocal microscopy (Olympus) images of 20 magnification of each lateral cerebellar section and transverse sections of the heart and analyze with ImageJ (National Institutes of Health, Bethesda, MD).

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2. Convert all photomicrographs to gray scale, select blank control images as background, and then subsequently use these to subtract the background from all images. 3. Use the same threshold for all images, and quantify the staining intensity of each section as the average optical density readings of four randomly selected areas within that section. 4. The final staining intensity of each group is calculated as the average of each staining intensity per section. 3.9 Statistical Analysis

1. Express all data as mean  SEM, and use two-way ANOVA followed by Bonferroni’s test as statistically evaluated (GraphPad version 5.01). 2. Use Student’s t-tests to determine and compare the effect of TGI stroke NHP versus sham NHP. 3. Use the Pearson correlation test to compare the alterations of volume densities of Purkinje cells and TUNEL+ staining intensity. 4. Consider significant at p < 0.05 of all comparisons.

4

Notes 1. Consult with your institution’s IACUC and veterinarian for appropriate dosage of anesthesia and analgesia. 2. Cerebral blood flow can be used to determine the success of the surgery during occlusion of the arteries. 3. Effective aseptic surgery practices minimize infections and other complications. 4. All animals are housed under normal conditions (20  C, 50% relative humidity, and a 12 h light/dark cycle). 5. Harvested tissue can be stored long-term in 30% sucrose solution. It is recommended to change the sucrose solution every 2 weeks to prevent fungal/bacterial growth. 6. TNF-α is used to identify the pro-inflammatory cytokine tumor necrosis factor-alpha. GFAP is used to identify astrocyte cells. HLA-DR (human leukocyte antigen) is used to identify major histocompatibility complex-presenting cells. 7. Alternative nuclear immunostaining, such as DAPI, can be used as substitute. 8. Section thickness can be between 20.0 and 21.0 microns after dehydration, which does not significantly alter the histopathological outcomes.

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References 1. Neubuerger KT (1954) Lesions of the human brain following circulatory arrest. J Neuropathol Exp Neurol 13(1):144–160 PubMed: 13118380 2. Fukuda S, del Zoppo GJ (2003) Models of focal cerebral ischemia in the nonhuman primate. ILAR J 44(2):96–104 PubMed: 12652004 3. Nemoto EM, Bleyaert AL, Stezoski SW, Moossy J, Rao GR, Safar P (1977) Global brain ischemia: a reproducible monkey model. Stroke 8(5):558–564 PubMed: 410121 4. Tabuchi E, Endo S, Ono T, Nishijo H, Kuze S, Kogure K (1992) Hippocampal neuronal damage after transient forebrain ischemia in monkeys. Brain Res Bull 29(5):685–690 PubMed: 1422866 5. Hara K, Yasuhara T, Matsukawa N, Maki M, Masuda T, Yu G, Xu L, Tambrallo L, Rodriguez NA, Stern DM, Kawase T, Yamashima T, Buccafusco JJ, Hess DC, Borlongan CV (2007) Hippocampal CA1 cell loss in a non-human primate model of transient global ischemia: a pilot study. Brain Res Bull 74 (1–3):164–171. https://doi.org/10.1016/j. brainresbull.2007.06.014 PubMed: 17683803 6. Hong JH, Lee H, Lee SR (2016) Protective effect of resveratrol against neuronal damage following transient global cerebral ischemia in mice. J Nutr Biochem 27:146–152. https:// doi.org/10.1016/j.jnutbio.2015.08.029 PubMed: 26421359 7. Ishikawa H, Tajiri N, Vasconcellos J, Kaneko Y, Mimura O, Dezawa M, Borlongan CV (2013) Ischemic stroke brain sends indirect cell death signals to the heart. Stroke 44 (11):3175–3182. https://doi.org/10.1161/ STROKEAHA.113.001714 PubMed: 24008571 8. Pearce A, Lockwood C, Van Den Heuvel C (2015) The use of therapeutic magnesium for neuroprotection during global cerebral ischemia associated with cardiac arrest and cardiac bypass surgery in adults: a systematic review protocol. JBI Database System Rev Implement Rep 13(4):3–13. https://doi.org/10.11124/ jbisrir-2015-1675 [PubMed: 26447069] 9. Sun L, Ai J, Wang N, Zhang R, Li J, Zhang T, Wu W, Hang P, Lu Y, Yang B (2010) Cerebral ischemia elicits aberration in myocardium contractile function and intracellular calcium handling. Cell Physiol Biochem 26 (3):421–430. https://doi.org/10.1159/ 000320584 PubMed: 20798527

10. Wang R, Liu YY, Liu XY, Jia SW, Zhao J, Cui D, Wang L (2014) Resveratrol protects neurons and the myocardium by reducing oxidative stress and ameliorating mitochondria damage in a cerebral ischemia rat model. Cell Physiol Biochem 34(3):854–864. https://doi. org/10.1159/000366304 PubMed: 25199673 11. Oppenheimer SM, Hachinski VC (1992) The cardiac consequences of stroke. Neurol Clin 10 (1):167–176 PubMed: 1557001 12. Prosser J, MacGregor L, Lees KR, Diener HC, Hacke W, Davis S, Investigators V (2007) Predictors of early cardiac morbidity and mortality after ischemic stroke. Stroke 38 (8):2295–2302. https://doi.org/10.1161/ STROKEAHA.106.471813 PubMed: 17569877 13. Bunevicius A, Kazlauskas H, Raskauskiene N, Mickuviene N, Ndreu R, Corsano E, Bunevicius R (2015) Role of N-terminal pro-B-type natriuretic peptide, high-sensitivity C-reactive protein, and inteleukin-6 in predicting a poor outcome after a stroke. Neuroimmunomodulation 22(6):365–372. https://doi.org/10. 1159/000381218 PubMed: 25967464 14. Sun YP, Wei CP, Ma SC, Zhang YF, Qiao LY, Li DH, Shan RB (2015) Effect of carvedilol on serum heart-type fatty acid-binding protein, brain natriuretic peptide, and cardiac function in patients with chronic heart failure. J Cardiovasc Pharmacol 65(5):480–484. https://doi. org/10.1097/FJC.0000000000000217 PubMed: 25945865 15. Shibazaki K, Kimura K, Aoki J, Sakai K, Saji N, Uemura J (2014) Plasma brain natriuretic peptide as a predictive marker of early recurrent stroke in cardioembolic stroke patients. J Stroke Cerebrovasc Dis 23(10):2635–2640. https://doi.org/10.1016/j. jstrokecerebrovasdis.2014.06.003 PubMed: 25238924 16. Kannel WB (2000) The Framingham study: ITS 50-year legacy and future promise. J Atheroscler Thromb 6(2):60–66 PubMed: 10872616 17. Lawlor DA, Smith GD, Leon DA, Sterne JA, Ebrahim S (2002) Secular trends in mortality by stroke subtype in the 20th century: a retrospective analysis. Lancet 360 (9348):1818–1823. https://doi.org/10. 1016/S0140-6736(02)11769-7 PubMed: 12480358 18. Lo EH, Dalkara T, Moskowitz MA (2003) Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci 4(5):399–415.

Histopathological Protocols for Characterizing Ischemic Secondary Cell Death https://doi.org/10.1038/nrn1106 PubMed: 12728267 19. Zhang ZG, Chopp M (2009) Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic. Lancet Neurol 8 (5):491–500. https://doi.org/10.1016/ S1474-4422(09)70061-4. PubMed: 19375666 20. Shibazaki K, Kimura K, Okada Y, Iguchi Y, Terasawa Y, Aoki J (2009) Heart failure may be associated with the onset of ischemic stroke with atrial fibrillation: a brain natriuretic peptide study. J Neurol Sci 281(1–2):55–57. https://doi.org/10.1016/j.jns.2009.02.374 PubMed: 19321180 21. Palumbo I, Palumbo B, Fravolini ML, Marcantonini M, Perrucci E, Latini ME, Falcinelli L, Sabalich I, Tranfaglia C, Schillaci G, Mannarino E, Aristei C (2016) Brain natriuretic peptide as a cardiac marker of

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transient radiotherapy-related damage in leftsided breast cancer patients: a prospective study. Breast 25:45–50. https://doi.org/10. 1016/j.breast.2015.10.004 PubMed: 26547836 22. Favilla CG, Ingala E, Jara J, Fessler E, Cucchiara B, Messe SR, Mullen MT, Prasad A, Siegler J, Hutchinson MD, Kasner SE (2015) Predictors of finding occult atrial fibrillation after cryptogenic stroke. Stroke 46 (5):1210–1215. https://doi.org/10.1161/ STROKEAHA.114.007763 PubMed: 25851771 23. Acosta SA, Mashkouri S, Nwokoye D, Lee JY, Borlongan CV (2017) Chronic inflammation and apoptosis propagate in ischemic cerebellum and heart of non-human primates. Oncotarget 8(61):102820–102834. https://doi. org/10.18632/oncotarget.18330 PubMed: 5732692

Chapter 18 Method for Stimulation of Hippocampal Neurogenesis by Transient Microneedle Insertion Shijie Song, Xiaoyung Kong, and Juan Sanchez-Ramos Abstract Brief stereotaxic insertion and removal of a microneedle into the hippocampus of mice result in stimulation of hippocampal neurogenesis. This approach has been previously applied to a mouse model of Alzheimer’s disease (Song et al., Cell Transplant 25:1853–1861, 2016). Further studies of fundamental cellular mechanisms of the brain’s response to micro-injury will be useful for investigation of potential neuroprotective and deleterious effects of targeted microlesions and deep brain stimulation in neurodegenerative diseases. Key words Focal brain injury, Brain repair, Astrocytosis, Microgliosis, Neurogenesis, Cytokines

1

Introduction Microneedle stimulation (acupuncture) has been used to relieve pain in China for millennia. Many diseases of the central nervous system (CNS) could not be treated by this method because of inaccessibility of the brain to a microneedle. In the last decade, deep brain stimulation (DBS) through chronically implanted metal electrodes into specific brain regions has become a common therapeutic choice for refractory movement disorders such as Parkinson’s disease (PD), tremors, and dystonia [1–6]. More recently, DBS has been applied to psychiatric [7] and behavioral disorders including depression [8], obsessive–compulsive disorder (OCD) [9], addiction, and, most recently, for disorders of consciousness. Long-term implantation of a fine metal electrode, even without chronic electrical stimulation, may produce unwanted effects, such as mild to severe inflammatory responses, micro-hemorrhages, and infections. Neuropathological examination of brain tissue from patients with DBS revealed activated astrocytes and microglia regardless of the disease. Electrical stimulation is not required to see signs of neuroinflammation; inflammatory changes have been observed around recording electrodes used for characterizing

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8_18, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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epileptogenic tissue and around cerebral spinal fluid (CSF) shunt catheters. Earlier studies of “stab injuries,” in which a foreign object (usually a metal) was inserted into rodent brain and immediately removed, had pointed to the importance of chemokine expression in mediating microgliosis and astrocytosis [10–13]. The acute and subacute reactions to transient implantation of a metal microneedle in the hippocampus were recently reported [14–18]. The needle insertion triggered a robust cellular response characterized by proliferation of microglia and astrocytes and resulted in stimulation of neurogenesis in the subgranular zone (SGZ) of the hippocampus [19–23]. Here we present detailed methods used to trigger hippocampal neurogenesis in rodents.

2

Materials

2.1

Animals

2.2

Equipment

C57BL6/J mice (Jackson Laboratory, Bar Harbor, ME USA). 1. Stereotaxic equipment (David Kopf Instruments, Tujunga, CA, USA). 2. BX6 fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) or LSM 510 Confocal Microscope (Zeiss, Oberkochen, Germany). 3. Digital camera system (DP-70, Olympus, Tokyo, Japan). 4. Dental drill (Cat. No. 58610, Corning Incorporated, Corning, NY14831, USA). 5. Stereotaxic needle (Lot. 3082804, Size No. 3(0.20)
40 mm, Seirin Corporation, Shizuoka, Japan). 6. Scalpel (Cat. No. 500236, World Precision Instruments, Sarasota, FL, USA). 7. Surgical blades (Cat. No. 500240, World Precision Instruments, FL, USA). 8. Tissue retractors (Stoelting Co. Wood Dale, IL, USA). 9. Thermal blanket pad (Fisher Scientific, Waltham, MA, USA). 10. Rectal thermometer (Fisher Scientific, Waltham, MA, USA). 11. Syringe (0.5–1 ml) (Fisher Scientific, Waltham, MA, USA). 12. Needles (22–24 gauge) (Fisher Scientific, Waltham, MA, USA). 13. Aluminum foil (Fisher Scientific, Waltham, MA, USA). 14. Cryostat (Leica, Wetzlar, Germany). 15. Cover glass (Cat. No. 12-548-5M, Fisher Scientific, Waltham, MA, USA).

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2.3 Drugs and Reagents

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1. Buprenorphine (Cat. No. 2808/10, Tocris, Minneapolis, MN, USA). 2. Ketamine (Cat. No. 3131/50, Tocris, Minneapolis, MN, USA). 3. Xylazine (Cat. No. 4963/10, Tocris, Minneapolis, MN, USA). 4. Bone wax (Sigma-Aldrich, St. Louis, MO, USA). 5. 5-Bromo-20 -deoxyuridine (BrdU) (Sigma-Aldrich, St. Louis, MO, USA). 6. Paraformaldehyde (Lot: 2856C348, Affymetrix, Inc. Cleveland, Ohio, USA). 7. 0.9% saline (Sigma-Aldrich, St. Louis, MO, USA). 8. Sucrose (Sigma-Aldrich, St. Louis, MO, USA). 9. Isopentane (Cat. N0. 270342-2L Sigma-Aldrich, St. Louis, MO, USA). 10. Normal goat or donkey serum (Vector Laboratories, Burlingame, CA, USA). 11. Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). 12. Phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA). 13. Primary antibodies: (a) Iba-1(catalog No. 019-19741, Wako, Osaka, Japan).

RRID:nlx_152487,

(b) GFAP (catalog No. 60-0032-7, RRID:AB_11203520, BioGenex, San Francisco, CA, USA). (c) DCX (catalog No. 18723, RRID:nlx_152244, Abcam, Cambridge, MA, USA). (d) Neu-N 1(catalog No. MAB377, Millipore, Burlington, MA, USA). (e) BrdU (catalog No. MCA2060, AbD S erotec, Raleigh, NC, USA). 14. Secondary antibodies: (a) Alexa Fluor 488 and 555 anti-goat IgG secondary antibodies (Molecular Probes/Invitrogen, Carlsbad, CA, USA).

3

Methods

3.1 Surgery and Microlesion

Animals undergo right hippocampus micro-lesioning using methods we have previously described [14, 15]. 1. Pretreat mice with buprenorphine (0.05 mg/kg, subcutaneously) at the time of anesthesia induction.

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Fig. 1 The Injection needle. Needle diameter, 200 μm; length, 40 mm

2. Inject ketamine 110 mg/kg + xylazine 11 mg/kg, intraperitoneally to induce anesthesia. 3. Check for deep anesthesia by assessing pain reflexes by interdigital pinching of the paw. If reflex is still present, animal is not deeply anesthetized. 4. The anesthetized mouse is fixed into a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). 5. Make a small incision with a scalpel, followed by retraction of scalp to expose the skull. 6. Drill two small holes (1 mm aperture) with a dental drill through the skull over the right hippocampus coordinates: (a) 1.8 mm anteroposterior and +1.0 mm mediolateral to bregma; the stereotaxic needle is lowered to a 2 mm depth and removed immediately. (b) 3.2 mm anteroposterior and +2.5 mm mediolateral to bregma; the lesioning needle is lowered to depth of 3.0 mm and removed immediately (Fig. 1). 7. Bone wax is used to cover the skull holes, and the skin incision is then sutured [14, 15]. 8. A computer-operated thermal blanket pad and a rectal thermometer are used to maintain body temperature within normal limits. 9. All animals are closely monitored until recovery from anesthesia and over the next 3 consecutive days. 10. Inject BrdU (Sigma-Aldrich, St. Louis, MO, USA) 100 μg/kg, subcutaneously bid for 3 days after inducing the injury (see Note 1).

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3.2 Immunohistology and Quantitative Image Analyses

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1. Mice are anesthetized with 150 mg/kg ketamine and 15 mg/ kg xylazine intraperitoneally and then transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde. 2. Brains are stored in 4% paraformaldehyde and transferred to 25% sucrose solution in 4% paraformaldehyde until they sink to the bottom. 3. Brains are then immersed in cold isopentane (cooled on dry ice) for 20 s, removed, placed on a small piece of aluminum foil resting on powdered dry ice for 1–2 min (to allow the isopentane evaporate), and finally wrapped in the foil and stored at 80  C until sectioning. 4. Brain slices are cut 30 μm thick on a cryostat (Leica, Wetzlar, Germany) set to 25  C. 5. Every sixth coronal section is taken from the hippocampus 2.0 mm in the anterior-posterior direction (from bregma 1.50 mm to bregma 3.50). Every sixth section is kept for immunostaining. 6. Selective immunostaining of astrocytes, microglia, and immature and mature neurons is performed with antibodies to GFAP, Iba-1, DCX, NeuN, and BrdU, respectively (see Note 3). 7. Brain sections are preincubated in phosphate-buffered saline (PBS) containing 10% normal serum (goat or donkey; Vector Laboratories, Burlingame, CA) and 0.3% Triton X-100 (Sigma, St. Louis, MO) for 30 min. 8. The sections are then transferred to a solution containing primary antibodies in 1% normal serum and 0.3% Triton X-100/PBS and incubated overnight at 4 ˚C.

3.3 Estimates of Hippocampal Neurogenesis

1. Labeled cells, double-stained with antibodies to BrdU and with markers of neural progenitor cells (nestin), immature neurons (DCX), and mature neurons (NeuN) are visualized with fluorescence microscopy using appropriate filters or with a Zeiss LSM 510 confocal fluorescence microscope (Fig. 2). 2. Unbiased estimates of the number of doubly labeled BrdU+ cells in the dentate gyrus are made by counting serially sectioned hippocampus according to the method previously described [24, 25] (see Note 2 for details). 3. Positively labeled cells are counted in every sixth section (30 μm thick) of tissue (180 μm). 4. The specific antibodies used for immunostaining and their sources are listed (see Note 3). 5. After incubation with primary antibody (anti-GFAP, antiNeuN, anti-BrdU, anti-Iba1, or anti-DCX), sections are

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Fig. 2 Microlesion stimulates neurogenesis. (a) Merged image of DCX and BrdU-immunoreactive cells on the unlesioned control side (2 weeks after the lesion). (b) Lesioned side illustrates increased DCX and BrdU (merged image). (c) The same as panel (b) but magnified; scale μm. Doublecortin (DCX)-immunoreactive cells in the subgranular zone of the dentate gyrus extend processes into the granular zone. The box inserted in (c) depicts confocal images of double-labeled DCX/BrdU cell at a higher power. Upper two panels are isolated for DCX (green) and BrdU (red) immunofluorescence, and the lower panel is the merged image (μm). (d) Summary data of DCX signal expressed as percent of DG field. Lesioned side exhibits a significantly increased DCX signal compared to control at both 2 and 4 weeks after the microlesion. Unlike microgliosis and astrocytosis, the DCX signal does not decline after 4 weeks. (e) Cell counts of double-labeled immature neurons (DCX/BrdU) born within 2 days of lesion placement. The lesion significantly increased birth of new neurons compared to unlesioned control side. However, the number of double-labeled cells was significantly less at 4 weeks than observed at 2 weeks

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washed and incubated for 1 h with fluorescently tagged secondary antibodies at room temperature (see Note 3). 6. Sections are then rinsed in PBS three times and covered with a cover glass. Green fluorescence signals from the labeled cells are visualized by fluorescence microscopy with appropriate filters. 7. All images are acquired and stored using an Olympus BX60 microscope with an attached digital camera system (DP-70, Olympus, Tokyo, Japan) allowing unbiased observers to independently count the double-labeled cells.

4

Notes 1. Preparation of fresh BrdU solution to label newborn neural cells, mice received 5-bromo-2-deoxyuridine (BrdU) (Sigma) injections (100 mg/kg i.p. bid, immediately after the surgery and 2 days after surgery) to label nascent cells during a 3-day period. 2. A modification to the optical dissector method was used so that cells on the upper and lower planes were not counted to avoid counting partial cells. The number of BrdU+cells counted in every sixth section was multiplied by six to get the total number of BrdU+cells in the dentate gyrus. For the quantification of double-labeled cells using immunofluorescence, the number of BrdU+ and BrdU+NeuN+ labeled cells is estimated using every 12th section taken throughout the dentate gyrus. Positive labeling is verified by confocal microscopy (Zeiss LSM 510). Cells determined to be BrdU+ and BrdU+NeuN+ positive are tallied and multiplied by the number of intervening sections. 3. Antibodies: Rabbit anti-Iba-1(1:500 containing 1:100 normal serum in PBS, catalog no. 019-19741, RRID:nlx_152487, Wako, Osaka, Japan); rabbit anti-GFAP (1:50 in PBS containing 1:100 normal serum without Triton X-100, catalog no. 60-0032-7, RRID:AB_11203520, BioGenex, San Francisco, CA); rabbit anti-DCX (1:1000 containing 1:100 normal serum, catalog no. 18723, RRID:nlx_152244, Abcam, Cambridge, MA); mouse anti-NeuN (1;100 containing 1:100 normal serum in PBS, catalog no. MAB377, Millipore, USA); rat anti-BrdU (1:100 containing 1:100 normal serum in PBS, catalog no. MCA2060, AbD Serotec, USA); and Alexa Fluor 488 and 555 anti-goat species-specific IgG diluted 1:400 in PBS (catalog no. A11070; RRID: AB_142134; Molecular Probes/Invitrogen, Carlsbad, CA).

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Acknowledgments Supported in part by VA Merit Grant and Ellis Research Fund. References 1. Chou KL, Forman MS, Trojanowski JQ, Hurtig HI, Baltuch GH (2004) Subthalamic nucleus deep brain stimulation in a patient with levodopa-responsive multiple system atrophy. Case report. J Neurosurg 100 (3):553–556 2. Flora ED, Perera CL, Cameron AL, Maddern GJ (2010) Deep brain stimulation for essential tremor: a systematic review. Mov Disord 25 (11):1550–1559 3. Krack P, Vercueil L (2001) Review of the functional surgical treatment of dystonia. Eur J Neurol 8(5):389–399 4. Stephan CL, Kepes JJ, SantaCruz K, Wilkinson SB, Fegley B, Osorio I (2001) Spectrum of clinical and histopathologic responses to intracranial electrodes: from multifocal aseptic meningitis to multifocal hypersensitivity-type meningovasculitis. Epilepsia 42(7):895–901 5. Vedam-Mai V, van Battum EY, Kamphuis W, Feenstra MG, Denys D, Reynolds BA, Okun MS, Hol EM (2012) Deep brain stimulation and the role of astrocytes. Mol Psychiatry 17 (2):124–131, 115 6. Vedam-Mai V, Yachnis A, Ullman M, Javedan SP, Okun MS (2012) Postmortem observation of collagenous lead tip region fibrosis as a rare complication of DBS. Mov Disord 27 (4):565–569 7. Marangell LB, Martinez M, Jurdi RA, Zboyan H (2007) Neurostimulation therapies in depression: a review of new modalities. Acta Psychiatr Scand 116(3):174–181 8. Henderson JM (2007) Vagal nerve stimulation versus deep brain stimulation for treatmentresistant depression: show me the data. Clin Neurosurg 54:88–90 9. de Koning PP, Figee M, van den Munckhof P, Schuurman PR, Denys D (2011) Current status of deep brain stimulation for obsessivecompulsive disorder: a clinical review of different targets. Curr Psychiatry Rep 13 (4):274–282 10. Ghirnikar RS, Lee YL, He TR, Eng LF (1996) Chemokine expression in rat stab wound brain injury. J Neurosci Res 46(6):727–733 11. Kaur C, Ling EA, Wong WC (1987) Origin and fate of neural macrophages in a stab wound of the brain of the young rat. J Anat 154:215–227

12. Song S, Kong X, Acosta S, Sava V, Borlongan C, Sanchez-Ramos J (2016) Granulocyte-colony stimulating factor promotes brain repair following traumatic brain injury by recruitment of microglia and increasing neurotrophic factor expression. Restor Neurol Neurosci 34(3):415–431 13. Song S, Kong X, Acosta S, Sava V, Borlongan C, Sanchez-Ramos J (2016) Granulocyte colony-stimulating factor promotes behavioral recovery in a mouse model of traumatic brain injury. J Neurosci Res 94 (5):409–423 14. Song S, Kong X, Sava V, Cao C, Acosta S, Borlongan C, Sanchez-Ramos J (2016) Transient Miro-needle insertion into hippocampus triggers neurogenesis and decreases amyloid burden in a mouse model of Alzheimer’s disease. Cell Transplant 25(10):1853–1861 15. Song S, Song S, Cao C, Lin X, Li K, Sava V, Sanchez-Ramos J (2013) Hippocampal neurogenesis and the brain repair response to brief stereotaxic insertion of a microneedle. Stem Cells Int 2013:205878 16. Sperling RA, Aisen PS, Beckett LA, Bennett DA, Craft S, Fagan AM, Iwatsubo T, Jack CR Jr, Kaye J, Montine TJ et al (2011) Toward defining the preclinical stages of Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 7 (3):280–292 17. Stone SS, Teixeira CM, Devito LM, Zaslavsky K, Josselyn SA, Lozano AM, Frankland PW (2011) Stimulation of entorhinal cortex promotes adult neurogenesis and facilitates spatial memory. J Neurosci 31 (38):13469–13484 18. Suthana N, Haneef Z, Stern J, Mukamel R, Behnke E, Knowlton B, Fried I (2012) Memory enhancement and deep-brain stimulation of the entorhinal area. N Engl J Med 366 (6):502–510 19. Laxton AW, Tang-Wai DF, McAndrews MP, Zumsteg D, Wennberg R, Keren R, Wherrett J, Naglie G, Hamani C, Smith GS, Lozano AM (2010) A phase I trial of deep brain stimulation of memory circuits in Alzheimer’s disease. Ann Neurol 68(4):521–534

Microneedle Insertion to Induce Neurogenesis 20. Patel NA, Song S, Cooper DR (2006) PKC delta alternatively spliced isoforms modulate cellular apoptosis in retinoic acid-induced differentiation of human NT2 cells and mouse embryonic stem cells. Gene Expr 13(2):73–84 21. Sanchez-Ramos J, Song S, Sava V, Catlow B, Lin X, Mori T, Cao C, Arendash GW (2009) Granulocyte colony stimulating factor decreases brain amyloid burden and reverses cognitive impairment in Alzheimer’s mice. Neuroscience 163(1):55–72 22. Shah SA, Schiff ND (2010) Central thalamic deep brain stimulation for cognitive neuromodulation - a review of proposed mechanisms and investigational studies. Eur J Neurosci 32 (7):1135–1144

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23. Song S, Wang X, Sava V, Weeber EJ, SanchezRamos J (2014) In vivo administration of granulocyte colony-stimulating factor restores long-term depression in hippocampal slices prepared from transgenic APP/PS1 mice. J Neurosci Res 92(8):975–980 24. Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (2001) Neurogenesis in the adult is involved in the formation of trace memories. Nature 410(6826):372–376 25. Shors TJ, Townsend DA, Zhao M, Kozorovitskiy Y, Gould E (2002) Neurogenesis may relate to some but not all types of hippocampal dependent learning. Hippocampus 12(5):578–584

INDEX A

D

Action potentials (APs)....... 14, 44, 75, 81, 86, 101, 103 Acupuncture .................................................................. 227 Adenosine triphosphate (ATP)........................81, 82, 161 Algorithms ............................................................ 188, 195 Allogenic....................................................................25, 34 Apoptosis ................................................................ 98, 161 Astrocytes .v, 1, 44, 60, 73–87, 162, 223, 227, 228, 231 Astrocytosis........................................................... 228, 232 ATP-linked respiration.................................................. 169

DAVID ........................................................ 148, 158, 159 Deep brain stimulation (DBS) ..................................... 227 Definitive NPCs .......................................... 28, 29, 32, 33 Depolarizations ............................................................... 75 Differentiated neurons (iNS) ...................................10, 15 Differentiation.........................v, vi, 1, 10, 12–16, 18, 22, 23, 28–30, 32, 33, 39, 43, 44, 46, 50, 51, 59, 62, 74, 89–94, 98, 100–104, 108, 111–115, 126, 164, 183, 187–196, 199–204 Direct conversion .....................................v, 10, 12, 14, 74 Directed differentiation ..................................... 10, 12–14 Dopamine-inducing factors (DIFs) ............................... 90 Dopamine-inducing factors-1 (DIF1) .................vi, 2, 91, 92, 121 Dopaminergic differentiation ................. 98, 99, 103, 108 Dopaminergic neuron differentiation .....................89–94, 101, 113, 126 Dopaminergic neurons ......................... vi, 14, 44, 89–94, 99, 102–104, 113, 119–127, 161, 165 Doublecortin ................................................................. 232 Drug screening................. vi, 90, 98, 104, 162, 167, 176 Dual inhibition of SMAD signaling.........................10, 98 Dual SMAD inhibition ......... 12, 13, 15, 16, 32, 33, 195

B Basal respiration ............................................................ 169 BBDuk ........................................................................... 147 Bone marrow cells (BMC)................................... 205–213 Bone morphogenetic protein (BMP) ................ 9, 13, 16, 32, 60, 61, 98, 99, 189, 192, 196 5-Bromo-20 -deoxyuridine (BrdU) ...................... 229–233

C C1 ................................. vi, 130, 131, 133–137, 140, 142 Calbindin ..................................................... 219, 220, 222 Calcium imaging ................................................... v, 73–87 Capacitance................................................................75, 85 Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) .............................................163, 167–169 Cavalieri estimator probe..................................... 218, 222 CD68 .................................................................... 218, 221 Cell death ............................................6, 39, 74, 215–223 Cell differentiation ................................ v, 29, 61, 64, 121 Cell reprogramming.................................................. v, 112 Cell transplantation.............vi, 37, 38, 98, 104, 205–213 Cellular bioenergetics ................................................... 161 Cerebellum ........................................................... 215–223 Chemically defined conditions ....................................... 60 Chemically directed differentiation................................ 44 CNS drugs ..................................................................... 119 Coating substrates.....................................................60, 61 Co-transfection ...................................207, 208, 211, 212 CRISPR/Cas9.......................................................... vi, 120 Cryopreservation............................................................. 44 Culture medium.............................. 28, 49, 55, 108, 188, 206–208, 211 Cytokines .............................................................. 210, 223

E Electrophysiology........................................ vi, 73–87, 101 Embryoid bodies (EBs) ............................. 10, 12, 15, 16, 20–22, 28–30, 126, 178, 180–182, 185, 194 Embryoid body formation........................................29, 59 Embryonic stem cells (ESCs) ............................. 1, 43, 59, 73, 90, 98, 112

F FASTQC............................................................... 146, 148 Fluidigm .........................................vi, 130, 131, 142, 147 Fluorescence-activated cell sorting (FACS).................. 61, 66, 69, 124, 125, 127, 130 Fluorescent calcium indicators ....................................... 74

G Gene expression ....................vi, 40, 44, 74, 99, 129, 194 GFAP ................................. 218, 220, 221, 223, 229, 231 Green fluorescent protein........................................ vi, 120

Marcel M. Daadi (ed.), Neural Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1919, https://doi.org/10.1007/978-1-4939-9007-8, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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238 Index

AND

PROTOCOLS

H

N

Heart.............................................................175, 215–223 Hematopoietic stem cell transplantation ............ 205–213 High-throughput screening ........................................... 44 Hippocampal neurogenesis ................................. 227–233 Hippocampus .........................................vii, 216, 228–231 HLA-DR.......................................................218, 221–223 Human induced pluripotent stem cells (hiPSC) ............vi, 97–115, 120, 121 Human pluripotent stem cells (hPSCs) ..............v, 10, 13, 15, 17, 25, 27, 29, 30, 32, 33, 49–51, 59–70, 176, 183, 194

NeuN .....................................................99, 229, 231, 233 Neural differentiation ...................... v, 10, 14, 15, 50, 99, 111, 112, 114, 115, 126, 188, 194, 195 Neural induction ....................................9, 10, 12, 13, 15, 16, 21, 27, 28, 30, 32, 44–46, 50, 59–62, 67, 68 Neural progenitor cells (NPCs) .......................... v, 13–15, 25, 29, 98, 99, 101, 231 Neural rosettes ........................................... 13, 21, 22, 26, 29, 33, 39, 59 Neural stem cells (NSCs).............................v–vii, 1–6, 16, 18, 20–22, 43–56, 59–70, 89–94, 98, 99, 119–127, 129–143, 145–159, 161, 165, 199–204 Neurodegenerative diseases .................... 26, 97, 161, 175 Neurogenesis .................................................. vii, 227–233 Neuroinflammation....................................................... 227 Neurons .......................... v, 1, 9, 25, 44, 60, 73, 89, 119, 162, 222, 231 Neurospheres............................... 4–6, 13, 29, 31, 40, 93, 126, 130, 134, 164, 172, 202, 203 Nextera XT ........................................................... 130, 137 Next-generation sequencing (NGS) ................... 129, 130 Nonhuman primate (NHP)............................. vi, 97–115, 175–196, 199, 223 Nonhuman primate iPSCs................................... 175–186 Non-mitochondrial respiration .................................... 169 Notch signaling .................................................. 28, 33, 40 Nucleofection .............................120, 121, 125, 127, 181

I Iba-1 ..................................................................... 229, 231 Induced differentiation ...................................... 10, 12, 14 Induced neural progenitor (iNPCs) ........................14, 74 Induced neural progenitors (iNPCs) ............................. 10 Induced pluripotent stem cells (iPSCs) ................. vi, 1–6, 25, 39, 59, 61, 67, 73, 89–94, 97–115, 119–127, 130, 145–159, 161, 162, 175–186, 199–204 Induced pluripotent stem cells (iPSCs) characterization ................................................. 179 Inflammation .......................................................... 38, 216 Input resistance .........................................................75, 81 Interleukin-3 ........................................................ 207, 210 Interleukin-6 ........................................................ 207, 210 In vitro modeling ..................................................... vi, 104

K

O

Kallisto ......................................................... 147, 149, 151 Kriks and colleagues’ protocol ....................................... 98

Oligodendrocytes .......... v, 1, 25, 39, 44, 60, 73, 74, 162 Oligomycin ...................................................163, 167–169 Oxygen consumption .......................................... 162, 168

L Lentiviral vectors ............................vi, 206–208, 211, 212 Library preparation .............................................. 129–143 Luripotent stem cells ...................................................... 25

M Marmoset iPSCs.........................180, 182, 185, 201, 203 Marmosets ............................ vi, 179–182, 185, 186, 188, 190–194, 196, 199–204 Matrigel ............................ 13, 15, 19, 21, 22, 26–28, 30, 32, 59, 60, 112 Maximal respiration ...................................................... 169 Membrane potential............................17, 75, 81, 83, 161 Microgliosis .......................................................... 228, 232 Microlesions ................................................ 229, 230, 232 Microneedle stimulation...................................... 227, 228 Mitochondrial functions .......................................... vi, 161 Mitochondrial respiration ..........161, 162, 165, 167, 168 Multipotency ....................................................26, 44, 205

P Parkinson’s disease (PD) ......................... 44, 89, 98, 119, 161, 172, 227 Parthenogenetic stem cell..................................... v, 43–56 Passive membrane currents............................................. 75 Patch-clamp recording................................ 75, 81, 83, 84 Pathway inhibition ............................................. 60, 61, 99 Perpetual NSC differentiation.......................................v, 1 Pluripotency .......................................v, vi, 10, 17, 33, 44, 51, 53, 60, 99, 183, 185, 192, 196 Pluripotent stem cells......................... 1–6, 43, 56, 59–70, 74, 89–94, 97–115, 175–188, 190, 194, 195 Proton leak .................................................................... 169 Purkinje cells...................................................14, 222, 223

R Regenerative medicine ..........................v, vi, 60, 175, 176 Reporter................................................................ 119–127

METHODS Reprogramming ......................................v, 10, 12, 14, 74, 75, 98, 112, 180 RNA sequencing (RNA-seq)....................vi, 44, 129, 145 Rodents.................................................................v, 33, 38, 39, 199, 216, 228 Rotenone .............................................163, 167–170, 172

S SCDE ............................................................148, 153–158 Scorecard ..................................................... 176, 183, 185 Seahorse assay................................................................ 165 Secondary injury .......................................................34, 38 Self-renewable neural stem cells ................................. vi, 4, 199–204 Self-renewal ................................................. 1, 33, 40, 185 Single-cell analysis ................................................ 130, 146 Single-cell RNA-seq (scRNA-seq) ................ vi, 129, 130, 145–159 Single cells ......................................... vi, 5, 10, 30–33, 51, 55, 61, 69, 94, 108, 113, 124, 125, 129–143, 145–159, 201, 212 Singular.................................................................. 80, 147, 151–153, 159 Sleuth ............................................................147, 149–151 Small molecules ........................... v, vi, 10, 18, 19, 22, 44, 60–62, 90, 119, 165, 188, 189, 192, 193, 196 SMART-Seq................................................. 130, 134, 135 Sonic hedgehog (SHH)............................... 9, 14, 90, 98, 99, 105, 106, 109–111, 113 Spinal cord injury (SCI) .......................................v, 33, 44

AND

PROTOCOLS: NEURAL STEM CELLS Index 239

Stem cells ..................................v, 1–6, 16, 18, 20–22, 25, 43–56, 59–70, 74, 75, 89–94, 97–115, 119–127, 129–143, 145–159, 161, 175–188, 190, 194, 195, 199–213 Synaptic inputs ................................................................ 75

T Three dimensional neural differentiations ...............14, 15 TNF-α ..................................................218, 220, 221, 223 Transient global ischemia (TGI) ......................... 215–223 Transplant ........................................................... 34, 38, 39 Transplantation ..................................... vi, 33, 37, 55, 94, 98, 99, 104, 203, 205–213 Tripotent.......................................................................... 25 Tyrosine hydroxylase (TH) .......................vi, 91, 99, 102, 119, 123, 126, 127

U Unfertilized oocytes........................................................ 43

V Viral transduction................................................. 205, 208

W Whole-cell patch clamp............................. 81, 83, 84, 101 Wnt ......................... 9, 13, 14, 60, 90, 98, 190, 192, 193

X Xenogenic ........................................................... 34, 60, 61