Abstract

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This study, conducted as part of the IMI2 NeuroDeRisk initiative,evaluates spontaneous calcium (Ca²⁺) oscillations in 2D and 3D neuronalnetworks derived from human induced pluripotent stem cells (hiPSCs), comparingtheir responses to those from 2D mouse cortical neuron cultures. Using adefined set of compounds with known seizurogenic risk, the study assessednetwork-level Ca²⁺ activity to explore model predictivity for seizureliability. The findings show higher concordance between 3D hiPSC neurospheroidsand mouse cortical networks—particularly in detecting decreases in oscillationfrequency and amplitude—suggesting that neurospheroids offer a reproducible,human-relevant platform for neurotoxicity screening.

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Key Learnings

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• 3D hiPSC neurospheroids more closelyreplicate native mouse cortical Ca²⁺ response patterns than 2D hiPSC networks,particularly for compounds with known seizure risk.

• Decreases in Ca²⁺ oscillationfrequency and amplitude are more predictive of seizurogenicity than increases.

• Network maturation and dimensionalitysignificantly impact the reliability and interpretability of neurotoxicityassays.

• While 2D hiPSC models show highsensitivity, their specificity is limited due to variability and highfalse-positive rates.

• Neurospheroids demonstrated robust,reproducible responses across test sites and platforms, validating their use inhigh-throughput neurotoxicity assessments.

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Methods

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Models Used:

• 2D hiPSC-derived GlutaNeuron +astrocyte networks

• 3D hiPSC-derived neurospheroids (50%neurons, 50% astrocytes)

• Primary mouse cortical neurons (2D)

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Assay:

• Ca²⁺ oscillation imaging using FLIPRand FDSS platforms

• Quantification of peak frequency andamplitude in response to pharmacological compounds

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Compound Set:

• 26 compounds sourced from FAERS andliterature, categorized as seizurogenic or non-seizurogenic

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Analysis:

• Directional changes in Ca²⁺ frequencyand amplitude

• Concordance between models

• Contingency table analysis todetermine sensitivity, specificity, and Youden index for predictive value

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Conclusions

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• 3D neurospheroids showed the highestconcordance (77% for frequency, 65% for amplitude) with mouse cortical neuronsin identifying seizure risk.

• Combined decreases in Ca²⁺ frequencyand amplitude emerged as the most reliable predictors of seizurogenicpotential.

• 2D hiPSC models, while useful, showedvariable results due to shorter maturation times and greater variability.

• Neurospheroids offer a scalable,reproducible, and human-relevant alternative to animal models for preclinicalneurotoxicity screening.

• Further refinement in modelcomposition, maturation, and multi-parametric analysis will enhance predictiveaccuracy.