Molecular and Functional Characterization of Different BrainSphere Models for Use in Neurotoxicity Testing on Microelectrode Arrays

The currently accepted methods for neurotoxicity (NT) testing rely on animal studies. However, high costs and low testing throughput hinder their application for large numbers of chemicals. To overcome these limitations, in vitro methods are currently developed which are based on human induced pluripotent stem cells (hiPSC) that allow higher testing throughput at lower costs. We applied six different protocols to generate 3D BrainSphere models for acute NT evaluation. These include three different media for 2D neural induction and two media for subsequent 3D differentiation resulting in self-organized, organotypic neuron/astrocyte microtissues. All induction protocols yielded nearly 100 % nestin-positive hiPSC-derived neural progenitor cells (hiNPCs) yet with different gene expression profiles concerning regional patterning. Moreover, gene expression and immunocytochemistry analyses revealed that the choice of media determines neural differentiation patterns. On the functional level, BrainSpheres exhibited different levels of electrical activity on microelectrode arrays (MEA). Spike sorting allowed BrainSphere functional characterization with the mixed cultures consisting of GABAergic, glutamatergic, dopaminergic, serotonergic, and cholinergic neurons. A test method for acute NT testing, the human multi-neurotransmitter receptor (hMNR) assay, was proposed applying such MEA-based spike sorting. These models are not only promising tools in toxicology but also for drug development and disease modeling.

• Publication year

  2023

• Venue

  bioRxiv

• Publication date

  2023-03-13

• Fields of study

  Biology, Medicine, Engineering

• Identifiers
  DOI 10.3390/cells12091270PMID 37174670PMCID 10177384
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Poor Translatability of Biomedical Research Using Animals — A Narrative Review

  2023cited by this paper
• A human iPSC-based in vitro neural network formation assay to investigate neurodevelopmental toxicity of pesticides.

  2023cited by this paper
• Prolonged Differentiation of Neuron-Astrocyte Co-Cultures Results in Emergence of Dopaminergic Neurons

  2023cited by this paper
• Human IPSC 3D brain model as a tool to study chemical-induced dopaminergic neuronal toxicity

  2022cited by this paper
• Organophosphate insecticides disturb neuronal network development and function via non-AChE mediated mechanisms.

  2022cited by this paper
• Probabilistic Risk Assessment – The Keystone for the Future of Toxicology

  2022cited by this paper
• Multielectrode Arrays for Functional Phenotyping of Neurons from Induced Pluripotent Stem Cell Models of Neurodevelopmental Disorders

  2022cited by this paper
• Establishment of a human cell-based in vitro battery to assess developmental neurotoxicity hazard of chemicals.

  2022cited by this paper
• Scientific Validation of Human Neurosphere Assays for Developmental Neurotoxicity Evaluation

  2022cited by this paper
• Neuronal differentiation pathways and compound-induced developmental neurotoxicity in the human neural progenitor cell test (hNPT) revealed by RNA-seq

  2022cited by this paper
• Ready for regulatory use: NAMs and NGRA for chemical safety assurance.

  2022cited by this paper
• Current status and future directions for a neurotoxicity hazard assessment framework that integrates in silico approaches.

  2022cited by this paper
• Next-generation risk assessment of chemicals - Rolling out a human-centric testing strategy to drive 3R implementation: The RISK-HUNT3R project perspective.

  2022cited by this paper
• Upscaling biological complexity to boost neuronal and oligodendroglia maturation and improve in vitro developmental neurotoxicity (DNT) evaluation.

  2022cited by this paper
• Effects of the Selective Serotonin Reuptake Inhibitor Fluoxetine on Developing Neural Circuits in a Model of the Human Fetal Cortex

  2021cited by this paper
• Gene–Environment Interactions in Developmental Neurotoxicity: a Case Study of Synergy between Chlorpyrifos and CHD8 Knockout in Human BrainSpheres

  2021cited by this paper
• Safer chemicals using less animals: kick-off of the European ONTOX project.

  2021cited by this paper
• New approach methods supporting read-across: Two neurotoxicity AOP-based IATA case studies.

  2021cited by this paper
• Academic application of Good Cell Culture Practice for induced pluripotent stem cells.

  2021cited by this paper
• Novel test strategies for in vitro seizure liability assessment

  2021cited by this paper
• External Scientific Report on the Interpretation of Data from the Developmental Neurotoxicity In Vitro Testing Assays for Use in Integrated Approaches for Testing and Assessment

  2021cited by this paper
• Cholinergic control of striatal GABAergic microcircuits

  2021cited by this paper
• Comparison of Acute Effects of Neurotoxic Compounds on Network Activity in Human and Rodent Neural Cultures.

  2021cited by this paper
• Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro

  2021cited by this paper
• Acute effects of the imidacloprid metabolite desnitro-imidacloprid on human nACh receptors relevant for neuronal signaling

  2021cited by this paper
• Neurotoxic reactive astrocytes induce cell death via saturated lipids

  2021cited by this paper
• Measurement of of Differentiated Human iPSC-Derived Neurospheres Recorded by Microelectrode Arrays (MEA)

  2021cited by this paper
• Membrane trafficking and positioning of mGluRs at presynaptic and postsynaptic sites of excitatory synapses.

  2021cited by this paper
• Hexachloronaphthalene (HxCN) impairs the dopamine pathway in an in vitro model of PC12 cells.

  2021cited by this paper
• Paving the way for application of next generation risk assessment to safety decision-making for cosmetic ingredients

  2021cited by this paper
• Haloperidol bound D2 dopamine receptor structure inspired discovery of subtype selective ligands

  2020cited by this paper
• Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres Are Suitable for Neurotoxicity Screening

  2020cited by this paper
• Neural In Vitro Models for Studying Substances Acting on the Central Nervous System.

  2020cited by this paper
• Parkinson disease

  2020cited by this paper
• Dopamine Receptor Subtypes, Physiology and Pharmacology: New Ligands and Concepts in Schizophrenia

  2020cited by this paper
• Applicability of hiPSC-Derived Neuronal Cocultures and Rodent Primary Cortical Cultures for In Vitro Seizure Liability Assessment

  2020cited by this paper
• Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays

  2020cited by this paper
• New ideas for non-animal approaches to predict repeated-dose systemic toxicity: Report from an EPAA Blue Sky Workshop.

  2020cited by this paper
• Characterization and application of electrically active neuronal networks established from human induced pluripotent stem cell-derived neural progenitor cells for neurotoxicity evaluation.

  2020cited by this paper
• Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) acutely affect human α1β2γ2L GABAA receptor and spontaneous neuronal network function in vitro

  2020cited by this paper
• Incorporation of stem cell-derived astrocytes into neuronal organoids to allow neuro-glial interactions in toxicological studies.

  2020cited by this paper
• Haloperidol bound D2 dopamine receptor structure inspired the discovery of subtype selective ligands

  2020cited by this paper
• Antidepressant Paroxetine Exerts Developmental Neurotoxicity in an iPSC-Derived 3D Human Brain Model

  2020cited by this paper
• Human neuronal signaling and communication assays to assess functional neurotoxicity

  2020cited by this paper
• Toward a Global Understanding of Chemical Pollution: A First Comprehensive Analysis of National and Regional Chemical Inventories.

  2020cited by this paper
• Neural Stem Cells and Methods for Their Generation From Induced Pluripotent Stem Cells in vitro

  2020influential reference
• Suitability of 3D human brain spheroid models to distinguish toxic effects of gold and poly-lactic acid nanoparticles to assess biocompatibility for brain drug delivery

  2019cited by this paper
• Protein pathway analysis to study development-dependent effects of acute and repeated trimethyltin (TMT) treatments in 3D rat brain cell cultures.

  2019cited by this paper
• Effect fingerprints of antipsychotic drugs on neural networks in vitro

  2019cited by this paper
• Ontogenetic expression of thyroid hormone signaling genes: An in vitro and in vivo species comparison

  2019cited by this paper
• Towards animal-free neurotoxicity screening: Applicability of hiPSC-derived neuronal models for in vitro seizure liability assessment.

  2019cited by this paper
• Parkinson disease

  2019cited by this paper
• Toxicity testing in the 21st century: progress in the past decade and future perspectives

  2019cited by this paper
• Concentration–response evaluation of ToxCast compounds for multivariate activity patterns of neural network function

  2019cited by this paper
• Innovative models for in vitro detection of seizure.

  2019cited by this paper
• Functional characterization of human pluripotent stem cell-derived cortical networks differentiated on laminin-521 substrate: comparison to rat cortical cultures

  2019cited by this paper
• Group II Metabotropic Glutamate Receptors Mediate Presynaptic Inhibition of Excitatory Transmission in Pyramidal Neurons of the Human Cerebral Cortex

  2019cited by this paper
• Astrocytes Maintain Glutamate Homeostasis in the CNS by Controlling the Balance between Glutamate Uptake and Release

  2019cited by this paper
• Studying Human Neurological Disorders Using Induced Pluripotent Stem Cells: From 2D Monolayer to 3D Organoid and Blood Brain Barrier Models.

  2019cited by this paper
• Astrogliogenesis in human fetal brain: complex spatiotemporal immunoreactivity patterns of GFAP, S100, AQP4 and YKL‐40

  2019cited by this paper
• Robust Generation of Person-Specific, Synchronously Active Neuronal Networks Using Purely Isogenic Human iPSC-3D Neural Aggregate Cultures

  2019cited by this paper
• Adherent vs. Free-Floating Neural Induction by Dual SMAD Inhibition for Neurosphere Cultures Derived from Human Induced Pluripotent Stem Cells

  2018cited by this paper
• Functional and Mechanistic Neurotoxicity Profiling Using Human iPSC-Derived Neural 3D Cultures

  2018cited by this paper
• Effect of prolonged differentiation on functional maturation of human pluripotent stem cell-derived neuronal cultures.

  2018cited by this paper
• Patterning factors during neural progenitor induction determine regional identity and differentiation potential in vitro.

  2018cited by this paper
• Neurotoxic effects of organophosphorus pesticides and possible association with neurodegenerative diseases in man: A review.

  2018cited by this paper
• Microglia Increase Inflammatory Responses in iPSC-Derived Human BrainSpheres

  2018cited by this paper
• Accelerating the Pace of Chemical Risk Assessment.

  2018cited by this paper
• Human iPSC‐derived neuronal models for in vitro neurotoxicity assessment

  2018cited by this paper
• Rotenone exerts developmental neurotoxicity in a human brain spheroid model

  2018cited by this paper
• Literature review and appraisal on alternative neurotoxicity testing methods

  2018cited by this paper
• Drug discovery and development: Role of basic biological research

  2017cited by this paper
• A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells

  2017cited by this paper
• A multi‐laboratory evaluation of microelectrode array‐based measurements of neural network activity for acute neurotoxicity testing

  2017cited by this paper
• The antipsychotic drugs olanzapine and haloperidol modify network connectivity and spontaneous activity of neural networks in vitro

  2017cited by this paper
• 3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects

  2017cited by this paper
• Developmental excitatory-to-inhibitory GABA-polarity switch is disrupted in 22q11.2 deletion syndrome: a potential target for clinical therapeutics

  2017cited by this paper
• Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

  2017cited by this paper
• The Timing of the Excitatory-to-Inhibitory GABA Switch Is Regulated by the Oxytocin Receptor via KCC2

  2016influential reference
• Editor's Highlight: Evaluation of a Microelectrode Array-Based Assay for Neural Network Ontogeny Using Training Set Chemicals.

  2016cited by this paper
• Neural Subtype Specification from Human Pluripotent Stem Cells.

  2016cited by this paper
• Buspirone requires the intact nigrostriatal pathway to reduce the activity of the subthalamic nucleus via 5-HT1A receptors.

  2016cited by this paper
• Emamectin is a non-selective allosteric activator of nicotinic acetylcholine receptors and GABAA/C receptors.

  2016cited by this paper
• Induced pluripotent stem cell technology: a decade of progress

  2016cited by this paper
• Huntington disease: a single-gene degenerative disorder of the striatum

  2016cited by this paper
• A Human Brain Microphysiological System Derived from Induced Pluripotent Stem Cells to Study Neurological Diseases and Toxicity

  2016influential reference
• The Indispensable Roles of Microglia and Astrocytes during Brain Development

  2016cited by this paper
• Is the time right for in vitro neurotoxicity testing using human iPSC-derived neurons?

  2016cited by this paper
• Comparative neurotoxicity screening in human iPSC-derived neural stem cells, neurons and astrocytes.

  2016cited by this paper
• Chronic 14-day exposure to insecticides or methylmercury modulates neuronal activity in primary rat cortical cultures.

  2016cited by this paper
• An analysis of the attrition of drug candidates from four major pharmaceutical companies

  2015cited by this paper
• A LUHMES 3D dopaminergic neuronal model for neurotoxicity testing allowing long-term exposure and cellular resilience analysis

  2015cited by this paper
• Reversing excitatory GABAAR signaling restores synaptic plasticity and memory in a mouse model of Down syndrome

  2015cited by this paper
• Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells

  2015cited by this paper
• Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

  2015cited by this paper
• The GABAA Receptor as a Therapeutic Target for Neurodevelopmental Disorders.

  2015cited by this paper
• Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models.

  2015cited by this paper
• Molecular identity of human outer radial glia during cortical development.

  2015cited by this paper
• Multiparametric characterisation of neuronal network activity for in vitro agrochemical neurotoxicity assessment.

  2015cited by this paper
• Astrocytes Protect against Isoflurane Neurotoxicity by Buffering pro-brain–derived Neurotrophic Factor

  2015cited by this paper

• New approach methodologies (NAMs) for improved developmental and reproductive toxicity (DART) assessment with focus on endocrine disruption - a PARC project.

  2026cites this paper
• Modeling the developing nervous system: a neuroscience perspective on the use of NAMs in DNT testing.

  2025cites this paper
• Multi-behavioral phenotyping in early-life-stage zebrafish for identifying disruptors of non-associative learning

  2025influential citation
• New Approach Methods (NAMs) for genotoxicity assessment of nano- and advanced materials; Advantages and challenges.

  2025cites this paper
• Application of a high-density microelectrode array assay using a 3D human iPSC-derived brain microphysiological system model for in vitro neurotoxicity screening of environmental compounds

  2025cites this paper
• Multi-behavioral fingerprints can identify potential modes of action for neuroactive environmental chemicals.

  2025cites this paper
• 2024 International Academy of Toxicologic Pathology (IATP) Satellite Symposium: New Approach Methodologies (NAMs) for Neurotoxicity Assessment and Regulatory Perspectives

  2025cites this paper
• Development of a network formation assay for developmental neurotoxicity hazard screening using 3D human iPSC derived BrainSpheres

  2025influential citation
• Perfluorooctanesulfonic acid (PFOS) antagonizes gamma-aminobutyric acid (GABA) receptors in larval zebrafish and mammalian models

  2025influential citation
• CHIASMA: Advancing chemicals and materials safety and sustainability assessments through innovative integration of in vitro and in silico (new approach) methodologies

  2025cites this paper
• A scoping review of stem cell models of leukodystrophies: advances in understanding pathophysiological mechanisms

  2025cites this paper
• Developmental neurotoxicity evaluation of di(2-ethylhexyl) phthalate (DEHP) and three alternative plasticizers in human neurospheres.

  2025cites this paper
• New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project

  2024cites this paper
• Rigor and reproducibility in human brain organoid research: Where we are and where we need to go

  2024cites this paper
• 3D Spheroid and Organoid Models to Study Neuroinfection of RNA Viruses.

  2024cites this paper
• HiPSC-derived 3D neural models reveal neurodevelopmental pathomechanisms of the Cockayne Syndrome B

  2024influential citation
• A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids.

  2024cites this paper
• Advancing Endocrine Disruptors via In Vitro Evaluation: Recognizing the Significance of the Organization for Economic Co-Operation and Development and United States Environmental Protection Agency Guidelines, Embracing New Assessment Methods, and the Urgent Need for a Comprehensive Battery of Tests

  2024cites this paper
• Transcriptomic characterization of 2D and 3D human induced pluripotent stem cell-based in vitro models as New Approach Methodologies for developmental neurotoxicity testing.

  2024cites this paper
• State of the science on assessing developmental neurotoxicity using new approach methods.

  2024cites this paper
• Distribution, contribution and regulation of nestin+ cells

  2023cites this paper
• Multi-behavioral phenotyping in zebrafish identifies a novel disruptor of non-1 associative learning with environmental and human relevance 2

  year unknowncites this paper