ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:http://www.springer.com/series/7651

Cell-Based Assays UsingiPSCs for Drug Development

and Testing

Edited by

Carl-Fredrik Mandenius

Division of Biotechnology, Department of Physics, Chemistry and Biology (IFM), Linköping University,Linköping, Sweden

James A. Ross

Tissue Injury and Repair Group, University of Edinburgh, Edinburgh, UK

EditorsCarl-Fredrik MandeniusDivision of BiotechnologyDepartment of PhysicsChemistry and Biology (IFM)Linkoping UniversityLinkoping, Sweden

James A. RossTissue Injury and Repair GroupUniversity of EdinburghEdinburgh, UK

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-4939-9476-2 ISBN 978-1-4939-9477-9 (eBook)https://doi.org/10.1007/978-1-4939-9477-9

© Springer Science+Business Media, LLC, part of Springer Nature 2019This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon 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 regulationsand 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 tobe 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.

Cover Caption: Morphogenesis of hiPSC-derived endothelial cells. hiPSC derived endothelial cells (left image) formingmicro tubes in matrigel (right image). Live cells were stained with DRAQ5™ (red, nuclear) and /or calcein AM (green).[Palakkan, Nanda & Ross]

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of SpringerNature.The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Preface

In 2012 Shinya Yamanaka and John Gurdon were awarded the Nobel Prize for Physiologyor Medicine for their work demonstrating that mature cells can be reprogrammed to a stemcell phenotype. John Gurdon’s key achievement in 1962 provided the first experimentalevidence of reprogramming when he transplanted amphibian somatic cell nuclei into enu-cleated oocytes. This breakthrough work showed that each nucleus of a differentiated cellretains a complete set of genetic instructions for the whole organism. Shinya Yamanaka andhis team, inspired by this discovery and subsequent experimental work, demonstrated thatforced expression of four transcription factors could induce pluripotency in somatic cells.Subsequently, effective reprogramming methods in mouse cells (2006) and in human cells(2007) were developed. The reprogrammed cells were termed induced pluripotent stemcells (iPSCs). Since this seminal work there has been an explosion of research and develop-ment in the field of iPSC biology with reprogramming of somatic cells from different tissuesand from different disease genotypes together with a gradual increase in the expertiserequired to differentiate iPSCs toward specialized cell fates. This progress has led to theincreasing use of iPSCs for disease modeling, drug discovery, and safety.

The development of complex iPSC-derived cellular models and employing these modelsduring the process of drug discovery in order to identify the most efficacious drugs requiresenormous efforts that are difficult to accomplish within a single research institute orpharmaceutical company. For this reason, scientific consortia such as StemBANCC areinstrumental in fostering a close collaboration between academic groups, pharmaceuticalgroups, and the biotech industry. These large collaborative enterprises are founded oncomplementary scientific endeavors, drug discovery efforts, and toxicology expertise todevelop human iPSC-derived cellular models for drug discovery, efficacy, and safety pur-poses. A key area which required critical development was the development of differentia-tion protocols which could enable the production of key cell types from different organs andprovide the basis for disease modeling and drug development. Another key area was theneed to develop robust cell-based assays of particular utility in the drug developmentindustry but also of great utility to researchers interested in the biology of particular tissuesor cell types. The major goal of this book is to present a set of differentiation and assayprotocols useful to researchers involved in disease modeling, drug discovery, and the cellbiology of a variety of tissues.

The introductory chapter describes the requirements and preconditions for usinghuman induced pluripotent cell lines in assay development within the pharmaceuticalindustry. The subsequent chapters provide protocols for the bulk culture of iPSCs andiPSC-derived cardiomyocytes by bioreactor technologies and detailed protocols for thedifferentiation of iPSC-derived endoderm, hepatocytes, vascular endothelial cells, cardio-myocytes, renal podocytes, neurons, and astrocytes. There are detailed protocols for high-throughput metabolic and proteomic assays, high content assays of mitochondrial functionand autophagy, electrophysiology assays, and specialized lab-on-a-chip and fluorescentassays.

We are indebted to all the contributing authors for sharing their expertise, to ProfessorJohn M. Walker for inviting us to edit this volume of the MiMB series and his supportduring the preparation of the book, and to Dr. Patrick J. Marton and Professor Kursad

v

Turksen for their support. Finally, we are particularly indebted to the support received fromthe Innovative Medicines Initiative Joint Undertaking under grant no. 115439, resources ofwhich are composed of financial contribution from the European Union’s Seventh Frame-work Programme (FP7/2007-2013) and EFPIA companies.

Linkoping, Sweden Carl-Fredrik MandeniusEdinburgh, UK James A. Ross

vi Preface

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Cell-Based Assays Using Differentiated Human InducedPluripotent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Zameel Cader, Martin Graf, Mark Burcin, Carl-Fredrik Mandenius,and James A. Ross

PART I DIFFERENTIATION OF IPC TO TARGET CELLS FOR ASSAYS

2 Monolayer Generation of Vascular Endothelial Cellsfrom Human Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Klaus Christensen, Filip Roudnicky, Mark Burcin,and Christoph Patsch

3 Generating Human iPSC-Derived Astrocytes with ChemicallyDefined Medium for In Vitro Disease Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Katharina Janssen, Lamiaa Bahnassawy, Claudia Kiefer,Jurgen Korffmann, Georg C. Terstappen, Viktor Lakics,Miroslav Cik, and Peter Reinhardt

4 Human Induced Pluripotent Stem Cell-Derived DefinitiveEndoderm Bulk Culture and Hepatic Differentiation. . . . . . . . . . . . . . . . . . . . . . . . 41Anwar A. Palakkan, Jyoti Nanda, and James A. Ross

5 Production of Cardiomyocytes from Human PluripotentStem Cells by Bioreactor Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55Caroline Halloin, Michelle Coffee, Felix Manstein,and Robert Zweigerdt

6 Improved Protocol for Cardiac Differentiation and Maturationof Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71Imen Jebeniani, Shunli Ding, and Michel Puceat

7 Human Pluripotent Stem Cell Expansion in Stirred Tank Bioreactors . . . . . . . . . 79Felix Manstein, Caroline Halloin, and Robert Zweigerdt

8 A Protocol for One-Step Differentiation of Human InducedPluripotent Stem Cells into Mature Podocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93Cormac Murphy, Elisabeth Feifel, Paul Jennings,Gerhard Gstraunthaler, and Anja Wilmes

9 Differentiation of hiPS Cells into Definitive Endodermfor High-Throughput Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101Teresa Bluhmki, Stefanie Traub, Eva Schruf, James Garnett,Florian Gantner, Daniel Bischoff, and Ralf Heilker

vii

PART II HIGH THROUGHPUT METABOLOMICS AND PROTEOMIC

CELL-BASED ASSAYS

10 LC-MS/MS-Based Metabolomics for Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . 119Anna Artati, Cornelia Prehn, and Jerzy Adamski

11 Preparation of iPSCs for Targeted Proteomic Analysis . . . . . . . . . . . . . . . . . . . . . . . 131Anna Baud, Wendy E. Heywood, Daniel Little, Paul Gissen,and Kevin Mills

12 Measurement of Bile Acids as a Marker of the Functionalityof iPSC-Derived Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141Francesca Mazzacuva, Maelle Lorvellec, Agostino Cilibrizzi,Kevin Mills, Peter Clayton, and Paul Gissen

13 Mass Spectrometry Measurement of Albumin–Alpha FetoproteinRatio as an Indicator of iPSC-Derived Hepatocyte Differentiation . . . . . . . . . . . . 149Francesca Mazzacuva, Maelle Lorvellec, Agostino Cilibrizzi,Kevin Mills, Wendy E. Heywood, Peter Clayton, and Paul Gissen

PART III HIGH CONTENT IMAGING

14 Seeding Induced Pluripotent Stem Cell-Derived Neurons onto384-Well Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159Daniel Little, Christin Luft, Oliver Pezzini-Picart,Olukunbi Mosaku, Robin Ketteler, Michael J. Devine,and Paul Gissen

15 High-Content Autophagy Analysis in iPSC-Derived NeuronsUsing Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165Daniel Little, Christin Luft, Olukunbi Mosaku, Robin Ketteler,Michael J. Devine, and Paul Gissen

16 High-Content Analysis of Mitochondrial Function in iPSC-DerivedNeurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175Daniel Little, Christin Luft, Olukunbi Mosaku, Robin Ketteler,Michael J. Devine, and Paul Gissen

17 Evaluating the Effect of Drug Compounds on Cardiac SpheroidsUsing the Cardiac Cell Outgrowth Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185Jonas Christoffersson, Florian Meier, Henning Kempf,Kristin Schwanke, Michelle Coffee, Mario Beilmann,Robert Zweigerdt, and Carl-Fredrik Mandenius

PART IV ELECTROPHYSIOLOGY ASSAYS

18 Assay Procedures for Compound Testing of hiPSC-DerivedCardiomyocytes Using Multiwell Microelectrode Arrays . . . . . . . . . . . . . . . . . . . . . 197Udo Kraushaar and Elke Guenther

19 Multielectrode Array (MEA)-Based Detection of SpontaneousNetwork Activity in Human iPSC-Derived Cortical Neurons. . . . . . . . . . . . . . . . . 209Valeria Kizner, Sandra Fischer, and Maximilian Naujock

viii Contents

PART V MISCELLANEOUS CELL-BASED ASSAYS

20 Assessing Neuronal Excitability on a Fluorometric ImagingPlate Reader (FLIPR) Following a Defined Electrostimulation Paradigm . . . . . . 219Valeria Kizner, Sandra Fischer, Benjamin J€ahnke,and Maximilian Naujock

21 Fabrication of a Microfluidic Cell Culture Device UsingPhotolithographic and Soft Lithographic Techniques . . . . . . . . . . . . . . . . . . . . . . . 227Jonas Christoffersson and Carl-Fredrik Mandenius

22 Using a Microfluidic Device for Culture and Drug ToxicityTesting of 3D Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235Jonas Christoffersson and Carl-Fredrik Mandenius

23 hiPS Cell-Derived Neurons for High-Throughput Screening. . . . . . . . . . . . . . . . . 243Stefanie Traub and Ralf Heilker

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

Contents ix

Contributors

JERZY ADAMSKI � Research Unit Molecular Endocrinology and Metabolism, HelmholtzZentrum Munchen, German Research Center for Environmental Health (GmbH),Neuherberg, Germany; German Center for Diabetes Research (DZD e.V.), Neuherberg,Germany; Lehrstuhl fur Experimentelle Genetik, Technische Universit€at Munchen,Freising-Weihenstephan, Germany; Department of Biochemistry, Yong Loo Lin School ofMedicine, National University of Singapore, Singapore

ANNA ARTATI � Research Unit Molecular Endocrinology and Metabolism, HelmholtzZentrum Munchen, German Research Center for Environmental Health (GmbH),Neuherberg, Germany

LAMIAA BAHNASSAWY � Neuroscience Discovery, AbbVie Deutschland GmbH & Co KG,Ludwigshafen am Rhein, Germany

ANNA BAUD � Centre for Translational Omics, UCL Institute of Child Health, London, UKMARIO BEILMANN � Boehringer Ingelheim Pharma GmbH and Co. KG, Nonclinical Drug

Safety Germany, Biberach, GermanyDANIEL BISCHOFF � Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co.

KG, Biberach, GermanyTERESA BLUHMKI � Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co.

KG, Biberach, GermanyMARK BURCIN � Roche Pharmaceutical Research and Early Development, Roche Innovation

Center Basel, Basel, SwitzerlandZAMEEL CADER � Translational Molecular Neuroscience Group, Nuffield Department of

Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UKKLAUS CHRISTENSEN � Roche Pharmaceutical Research and Early Development, Roche

Innovation Center Basel, Basel, SwitzerlandJONAS CHRISTOFFERSSON � Division of Biotechnology, Department of Physics, Chemistry and

Biology (IFM), Linkoping University, Linkoping, SwedenMIROSLAV CIK � Neuroscience Discovery, AbbVie Deutschland GmbH & Co KG,

Ludwigshafen am Rhein, GermanyAGOSTINO CILIBRIZZI � Institute of Pharmaceutical Science, King’s College London, London,

UKPETER CLAYTON � Great Ormond Street Institute of Child Health, University College London,

London, UKMICHELLE COFFEE � Leibniz Research Laboratories for Biotechnology and Artificial Organs

(LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery,Hannover Medical School, Hannover, Germany; REBIRTH-Cluster of Excellence,Hannover Medical School, Hannover, Germany

MICHAEL J. DEVINE � MRC Laboratory for Molecular Cell Biology, University CollegeLondon, London, UK

SHUNLI DING � INSERM U1251 Universite Aix-Marseille, MMG, Marseille, FranceELISABETH FEIFEL � Division of Physiology, Medical University Innsbruck, Innsbruck, AustriaSANDRA FISCHER � CNS Diseases Research, Boehringer Ingelheim Pharma GmbH&Co. KG,

Biberach an der Riß, Germany

xi

FLORIAN GANTNER � Translational Medicine and Clinical Pharmacology, BoehringerIngelheim Pharma GmbH & Co. KG, Biberach, Germany

JAMES GARNETT � Immunology & Respiratory Diseases Research, Boehringer IngelheimPharma GmbH & Co. KG, Biberach, Germany

PAUL GISSEN � MRC Laboratory for Molecular Cell Biology, University College London,London, UK; Great Ormond Street Institute of Child Health, University College London,London, UK; NIHR Great Ormond Street Hospital Biomedical Research Centre,University College London, London, UK

MARTIN GRAF � Roche Pharmaceutical Research and Early Development, Roche InnovationCenter Basel, Basel, Switzerland

GERHARD GSTRAUNTHALER � Division of Physiology, Medical University Innsbruck, Innsbruck,Austria

ELKE GUENTHER � AIT Austrian Institute of Technology GmbH, Vienna, AustriaCAROLINE HALLOIN � Leibniz Research Laboratories for Biotechnology and Artificial Organs

(LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery,Hannover Medical School, Hannover, Germany; REBIRTH-Cluster of Excellence,Hannover Medical School, Hannover, Germany

RALF HEILKER � Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG,Biberach, Germany

WENDY E. HEYWOOD � Great Ormond Street Institute of Child Health, University CollegeLondon, London, UK; Centre for Translational Omics, UCL Institute of Child Health,London, UK

BENJAMIN J€AHNKE � CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co.KG, Biberach an der Riß, Germany

KATHARINA JANSSEN � Neuroscience Discovery, AbbVie Deutschland GmbH & Co KG,Ludwigshafen am Rhein, Germany; BG Neurochirurgie und Neurotraumatologie,Berufsgenossenschaftliches Universit€atsklinikum Bergmannsheil gGmbH, Bochum,Germany

IMEN JEBENIANI � INSERM U1251 Universite Aix-Marseille, MMG, Marseille, FrancePAUL JENNINGS � Division of Molecular and Computational Toxicology, Vrije Universiteit

Amsterdam, Amsterdam, The NetherlandsHENNING KEMPF � Leibniz Research Laboratories for Biotechnology and Artificial Organs

(LEBAO), Hannover Medical School, Hannover, GermanyROBIN KETTELER � MRC Laboratory for Molecular Cell Biology, University College London,

London, UKCLAUDIA KIEFER � Neuroscience Discovery, AbbVie Deutschland GmbH & Co KG,

Ludwigshafen am Rhein, GermanyVALERIA KIZNER � CNS Diseases Research, Boehringer Ingelheim Pharma GmbH& Co. KG,

Biberach an der Riß, GermanyJURGEN KORFFMANN � Neuroscience Discovery, AbbVie Deutschland GmbH & Co KG,

Ludwigshafen am Rhein, GermanyUDO KRAUSHAAR � Department of Electrophysiology, NMI Natural and Medical Sciences

Institute at the University of Tuebingen, Reutlingen, GermanyVIKTOR LAKICS � Neuroscience Discovery, AbbVie Deutschland GmbH & Co KG,

Ludwigshafen am Rhein, GermanyDANIEL LITTLE � MRC Laboratory for Molecular Cell Biology, University College London,

London, UK

xii Contributors

MAELLE LORVELLEC � MRC Laboratory for Molecular Cell Biology, University CollegeLondon, London, UK

CHRISTIN LUFT � MRC Laboratory for Molecular Cell Biology, University College London,London, UK

CARL-FREDRIK MANDENIUS � Division of Biotechnology, Department of Physics, Chemistryand Biology (IFM), Linkoping University, Linkoping, Sweden

FELIX MANSTEIN � Leibniz Research Laboratories for Biotechnology and Artificial Organs(LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery,Hannover Medical School, Hannover, Germany; REBIRTH-Cluster of Excellence,Hannover Medical School, Hannover, Germany

FRANCESCA MAZZACUVA � Mass Spectrometry Facility, King’s College London, London, UK;Great Ormond Street Institute of Child Health, University College London, London, UK

FLORIAN MEIER � Boehringer Ingelheim Pharma GmbH and Co. KG, Nonclinical DrugSafety Germany, Biberach, Germany

KEVIN MILLS � Great Ormond Street Institute of Child Health, University College London,London, UK; Centre for Translational Omics, UCL Institute of Child Health, London,UK

OLUKUNBI MOSAKU � MRC Laboratory for Molecular Cell Biology, University CollegeLondon, London, UK

CORMAC MURPHY � Division of Molecular and Computational Toxicology, Vrije UniversiteitAmsterdam, Amsterdam, The Netherlands

JYOTI NANDA � Tissue Injury and Repair Group, University of Edinburgh, Edinburgh, UKMAXIMILIAN NAUJOCK � CNSDiseases Research, Boehringer Ingelheim Pharma GmbH&Co.

KG, Biberach an der Riß, GermanyANWAR A. PALAKKAN � Tissue Injury and Repair Group, University of Edinburgh, Edinburgh,

UKCHRISTOPH PATSCH � Roche Pharmaceutical Research and Early Development, Roche

Innovation Center Basel, Basel, SwitzerlandOLIVER PEZZINI-PICART � MRC Laboratory for Molecular Cell Biology, University College

London, London, UKCORNELIA PREHN � Research Unit Molecular Endocrinology and Metabolism, Helmholtz

Zentrum Munchen, German Research Center for Environmental Health (GmbH),Neuherberg, Germany

MICHEL PUCEAT � INSERM U1251 Universite Aix-Marseille, MMG, Marseille, FrancePETER REINHARDT � Neuroscience Discovery, AbbVie Deutschland GmbH & Co KG,

Ludwigshafen am Rhein, GermanyJAMES A. ROSS � Tissue Injury and Repair Group, University of Edinburgh, Edinburgh, UKFILIP ROUDNICKY � Roche Pharmaceutical Research and Early Development, Roche

Innovation Center Basel, Basel, SwitzerlandEVA SCHRUF � Immunology & Respiratory Diseases Research, Boehringer Ingelheim Pharma

GmbH & Co. KG, Biberach, GermanyKRISTIN SCHWANKE � Leibniz Research Laboratories for Biotechnology and Artificial Organs

(LEBAO), Hannover Medical School, Hannover, GermanyGEORG C. TERSTAPPEN � Neuroscience Discovery, AbbVie Deutschland GmbH & Co KG,

Ludwigshafen am Rhein, Germany; OxStem Limited, Oxford, UKSTEFANIE TRAUB � Trenzyme GmbH, Konstanz, Germany

Contributors xiii

ANJA WILMES � Division of Molecular and Computational Toxicology, Vrije UniversiteitAmsterdam, Amsterdam, The Netherlands

ROBERT ZWEIGERDT � Leibniz Research Laboratories for Biotechnology and Artificial Organs(LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery,Hannover Medical School, Hannover, Germany; REBIRTH-Cluster of Excellence,Hannover Medical School, Hannover, Germany

xiv Contributors