Functional and Mechanistic Neurotoxicity Profiling Using Human iPSC-Derived Neural 3D Cultures

Abstract

Neurological disorders affect millions of people worldwide and appear to be on the rise. Whereas the reason for this increase remains unknown, environmental factors are a suspected contributor. Hence, there is an urgent need to develop more complex, biologically relevant, and predictive in vitro assays to screen larger sets of compounds with the potential for neurotoxicity. Here, we employed a human induced pluripotent stem cell (iPSC)-based 3D neural platform composed of mature cortical neurons and astrocytes as a model for this purpose. The iPSC-derived human 3D cortical neuron/astrocyte co-cultures (3D neural cultures) present spontaneous synchronized, readily detectable calcium oscillations. This advanced neural platform was optimized for high-throughput screening in 384-well plates and displays highly consistent, functional performance across different wells and plates. Characterization of oscillation profiles in 3D neural cultures was performed through multi-parametric analysis that included the calcium oscillation rate and peak width, amplitude, and waveform irregularities. Cellular and mitochondrial toxicity were assessed by high-content imaging. For assay characterization, we used a set of neuromodulators with known mechanisms of action. We then explored the neurotoxic profile of a library of 87 compounds that included pharmaceutical drugs, pesticides, flame retardants, and other chemicals. Our results demonstrated that 57% of the tested compounds exhibited effects in the assay. The compounds were then ranked according to their effective concentrations based on in vitro activity. Our results show that a human iPSC-derived 3D neural culture assay platform is a promising biologically relevant tool to assess the neurotoxic potential of drugs and environmental toxicants.

Figure 1.

Phenotypic 3D neural cultures assay development and implementation workflow.

Figure 2.

(A) Human iPSC-derived 3D neural culture spheroids, approximately 600 μm diameter, imaged with ImageXpress Micro Confocal system, 20x magnification, transmitted light. (B) Fluorescent images were taken after staining cells with marker-specific antibodies as described in Materials and Methods section. Images were taken using 20× objective in confocal mode. Spheroids are composed of a co-culture of active cortical neurons (identified by MAP2; green) and astrocytes (identified by GFAP; red). (C) Staining for Aquaporin 4 (green) showed the presence of mature GFAP-positive astrocytes. (D) Staining for Synapsin I (red) showed the presence of mature MAP2-positive neurons. B, C, and D show composite projection images of 3D neural cultures (30 images, 15 μm apart). Nuclei are stained with Hoechst 33342 (blue).

Figure 3.

(A) Gene expression profiling for neurotransmitters receptors. (B) Gene expression profiling for ion channels. Data presented for 3D neural cultures samples and adult brain samples. Both values were normalized to iPSC cells. KCNC1 and others represent Potassium Voltage-Gated Channels; SCN2A and other represent Sodium Voltage-Gated Channels; CACNA1D represent L-type calcium channels.

Figure 4.

(A) Representative spontaneous calcium oscillation signal traces shown for selected modulators of neuronal activity. Graphs display phenotypic responses of neuronal activity after exposure to neuromodulators. Unaffected regular Ca²⁺ oscillations patterns are presented as a control (DMSO, 0.15%). Traces are shown for 1 μM concentrations for all compounds with exception of haloperidol (0.1 μM) and dilantin (10 μM). (B) Neural spheroids respond to glutamate agonist and antagonist (kainic acid and CNQX, respectively); both show concentration-dependent responses according to the expected mechanism of action of the compound on the excitatory system. Kainic acid induces an excitotoxicity response at concentrations greater than 3 μM. (C) Three-dimensional neural cultures respond to the GABAergic agonists GABA and baclofen, with reduced peak count. (D) The anesthetic lidocaine (voltage-gated sodium channel blocker) and the antipsychotic medication haloperidol (dopamine D₂ receptor antagonist) show modulatory effects. Peak counts were measured 60 min after compound addition. Data represent averages of n = 3 replicates with standard deviation bars (haloperidol n = 2, kainic acid n = 4). Highest concentrations for some compounds not shown at the bar graphs (because peak count =0).

Figure 5.

Representative calcium oscillation signal traces for neurotoxic compounds representing different chemical classes: pesticides, flame retardants, and drugs. Shown are typical phenotypic responses including unaffected regular Ca²⁺ oscillations patterns (DMSO). Traces are shown for effective concentrations of compounds: 0.3 μM for rotenone; 1 μM for DDT and deltamethrin, 3 μM for diethylstilbestrol, and 10 μM for other compounds.

Figure 6.

Composite projection images of 3D neural cultures treated with 30 µM of indicated compounds for 24 h and then stained with a nuclear stain (Hoechst 33342), viability stain (Calcein AM), and mitochondria potential dye MitoTracker Orange CMTMRos for 2 h (2 µM, 1 µM, and 0.5 µM, respectively). Spheroids were imaged with the DAPI, FITC, and TRITC, 10× Plan Fluor objective, imaged using Z-stack of confocal images (30 images, 15 μm apart). Maximum projection images were analyzed using custom module editor for detection of spheroid size and shape, and also count of positive and negative cells in each tissue. The images show nuclei (blue), Calcein AM stain (green), and mitochondria (red).

Figure 7.

Assay variability for calcium oscillation and viability readouts. (A) Intra-plate variability of different measurements for the vehicle control samples within a representative plate, (DMSO) n = 24 after 24 h of incubation, data not normalized. Mean (black squares) is shown for each phenotype overlaid on top of gray circles representing individual well responses. Coefficients of variation (%CV) are shown for each phenotype. (B) Variabilities in vehicle controls for peak counts (per 10 min recording) measured for 3 representative plates. Mean is shown (black squares) for each of three experiments overlaid on top of gray circles representing individual well responses. %CVs are also shown for each experiment. (C) Comparison of compound responses (EC₅₀ values) between 2 plates for 8 test compounds (24 h treatment). Pearson correlation between experiments was r = 0.987 p < .0001, Spearman correlation r = 1.000, p < .0001.

Figure 8.

Heat map showing quantitative normalized concentration-response data for two selected measurements: the calcium oscillation peak counts (per 10 min) and cell viability for concentration range of 0.3–100 μM after 24 h of treatment. Responses at each time point and concentration are shown as a heat map (scale bar legend is on a side of the figure).

Figure 9.

(A) Compounds ranked according to the Bench Mark Concentrations (BMC) for peak count (per 10 min recording) values. Values for other calcium readout measurements are shown as different symbols. (B) Compounds were ranked according to their selectivity. Selectivity defined as > the square root of 10 (a difference of >0.5 in the log₁₀ of the BMC), based on the ratios between the lowest non-viability BMC (calcium oscillation read-outs) and BMC for viability (Calcein AM assay).

Figure 10.

Numbers of compounds from different categories tested positive or negative in the assay. The x-axis shows categories of compounds, and the y-axis shows the total number of compounds tested as positive/negative in the assay. The black bar indicates compounds that have previously been classified as developmental neurotoxicants (DNT) or neurotoxicants (NT) in the literature that were active (positive) in this assay; the dashed bar shows compounds that are classified as DNT/NT compounds in the literature that tested negative in this assay; the orange bar (the gray in back-white version) shows compounds with unknown toxicities that were positive and the white bar shows compounds with unknown toxicity in the literature which were tested as negative in the assay.