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. While 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.

Screening for modulators of neural network activity in 3D human iPSC-derived cortical spheroids

“…While not ruling out other sources, e.g. astrocytes, these data and that of others [19] provides strong evidence for a neuronal contribution to the whole neurospheroid Ca 2+ signal. Synchronous bursting of neurons in culture is believed to be a result of spontaneous miniature synaptic conductances in combination with random depolarizations that exert an intrinsic timing of bursting in the network.…”

A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids

“…Treatment with the known calcium channel blocker cobalt(II) chloride completely abolished these peaks (see Figure 4g ). Since the synchronous calcium activity of the AMOs and its modulation by inhibitors can be measured easily via HTS-friendly standard plate readers or specialized FLIPR Ca imagers ( Sirenko et al, 2019 ), AMOs may be a promising 3D model of human neural activity that allows directly assessing midbrain related organ functions in HTS.…”

“…While other groups have previously reported the use of human neural precursor cell (hNPC)-derived neurospheres for toxicity testing ( Moors et al, 2009 ), as well as a hiPSC-derived high-throughput-compatible spheroid model including the use of calcium oscillations as a readout to evaluate neurotoxicity ( Sirenko et al, 2019 ), these aggregates do not display structural tissue self-organization to the same extent as our organoids. Importantly, none of the available high-throughput-compatible protocols generates midbrain-specific organoids, but rather they focus on a cortical fate ( Sirenko et al, 2019 ). Our model of the human midbrain opens up the potential to perform 3D organoid-based HTS of midbrain-specific disorders including the highly prevalent Parkinson’s disease.…”

Assessment of ethanol-induced toxicity on iPSC-derived human dopaminergic neurons using a novel high-throughput mitochondrial neuronal health (MNH) assay

“…We suggest the MNH assay as a novel HT assay centered on human neurons that can be further multiplexed with additional imaging-based techniques, including for example the addition of an apoptotic dye to quantify cell death (35,36). iPSC-derived 3D neural cultures may also be employed to assess the neurotoxic potential of drugs and environmental toxicants (43). Therefore, future implementations should aim to adapt the MNH assay to 3D culture systems, such as iPSCderived brain organoids (44).…”