Screening for environmental toxins with a human brain in a dish model

In our daily lives, we are exposed to thousands of commercially used chemicals. Many of these chemicals are not toxic at typical exposure levels, but for thousands of chemicals, toxicological information is lacking. The National Academy of Sciences report on ‘‘Toxicity testing in the 21st century’’ highlighted the need for efficient methods to screen chemicals (e.g. insecticides) for their potential to cause toxicity. In this webinar, Dr. Lorena Saavedra (NeuCyte) discusses how measuring compound-induced changes to the spontaneous firing activity of human stem cell-derived neural cells in an MEA assay helped detect potentially harmful neurological side effects of compounds such as pyrethroid insecticides.


Audio Transcript: 

Thank you for joining us on today's coffee break webinar, today's topic is screening for environmental toxins with a human brain in a dish model. In our daily lives, we are exposed to thousands of commercially used chemicals many of these chemicals are not toxic at typical exposure levels but for thousands of chemicals toxicological information is lacking. The National Academy of Sciences report on toxicity testing in the 21st century highlighted the need for efficient methods to screen chemicals for their potential to cause toxicity.

One such example is insecticides, pyrethroid based insecticides are widely used for agricultural industrial, and residential pest control, although these compounds have been used for over 50 years in the United States their use has increased significantly in recent years, as a result of usage declines and other classes of insecticides.

Pyrethroids disrupt the kinetics of voltage-gated sodium channels in insect and mammalian neurons by prolonging the amount of time the channel is open, this alters neuronal activity and is the basis for the insecticidal and toxicological effects of pyrethroids. Exposure to high doses of pyrethroids in humans can cause hyperexcitability tremors, and involuntary and impaired movements.

When screening compounds for the potential to disrupt the nervous system, measuring neural activity is crucial since many neurotoxins are known to disrupt ion channel and receptor activity, furthermore these disruptions to neuronal excitability often precede or occur in the absence of other biochemical or morphological changes to the neuronal cells.

Today's presenter Dr. Lorena Saavedra is a principal scientist at NeuCyte, she will discuss how measuring drug-induced changes to the spontaneous firing activity of human stem cell-derived neural cells in MEA assays help detect potentially harmful neurological side effects of compounds, such as pyrethroid insecticides.

Dr. Saavedra is neuroscientist by training, she completed her doctoral studies at the University of Puerto Rico where she focused on deciphering several of the molecular aspects of learning and memory. Afterward, she continued to do a post-doctoral fellowship at Tufts University, where she studied the molecular and behavioral aspects of stress-related anxiety disorders, she currently is a principal scientist at NeuCyte where she has been developing human neural in vitro cell-based models for studying genetic-based neurological disorders and chemical-induced neurotoxicity testing.

Thank you for the great intro Melissa, as mentioned earlier I will be discussing the health of Cece assessment of chemicals using IPSC-derived neural cultures. The brain is the most complex organ of the body, with a hundred billion neurons, with different features being functionally integrated and organized in circuits and topological structures, a disturbance of the neuronal crosstalk in mature networks can have severe effects on individuals, including cognitive impairment and seizures. NeuCyte has developed an IPS cell-based human neural in vitro platform that reduces the complexity of the human brain while maintaining essential features of neuronal network activity.

Neucyte; SynFire neural cells are based on a direct conversion technology that allows for separately producing pure populations of different neuronal cell types. These can be individually cryopreserved as a modular system for plating accustomed numbers and ratios, for this presentation we will focus on a defined neural culture consisting of 70% excitatory glutamatergic neurons 30% inhibitory GABAergic neurons, and primary humanistic glial cells. 

SynFire based neural cultures have been highly characterized, they exhibit complex morphologies and express the correct neuronal markers they also possess the intrinsic electrophysiological properties and synaptic competence expected from mature neurons, such as fast action potential, spikes spontaneous activity, and functional excitatory and inhibitory synapses, this was assessed by conventional patch-clamp, in which traditionally focuses on one cell at a time.

A way of overcoming the single-cell analysis limitation and assessing neuronal activity in a quantitative and medium-throughput manner is Axion's multielectrode array MEA platform. Here neurons are plated on an electrode grid that measures local field potentials and derives multiple parameters to describe the electrical activity of the cell population. This technology is particularly suited for studying effects on network activity which can be regarded as a manner of evaluating multiple neuronal key features.

NeuCyte's SynFire neurons demonstrate rapid formation of coordinated network activity with highly synchronized bursting that allows the assessments of complex neuronal function. Combining Axion's MEA platform with Neucyte's technology enables advanced in vitro studies of neuronal activity in human cells. This opens avenues for multiple applications for neurotoxicity testing, as well as drug discovery programs. To follow I will talk about an ongoing project in collaboration with the Schaefer lab at the EPA to determine the applicability of our system for neural toxicity assessment and protection. For this purpose we tested a series of compounds with well-established neurotoxicity effects, this included three negative controls; amoxicillin, salicylic acid, and glyphosate, one positive control, tributyltin, which is cytotoxic to neurons. For GABAa receptor antagonists; bicuculline, lindane, PTX, and dieldrin, and 4 pyrethroids; Deltamethrin, permethrin cypermethrin and esfenvalerate, which causes effects on voltage-gated sodium channels.

We see that neural co cultures as described before, on Axion's Maestro system - and let the neurons mature to exhibit synchronous firing. On day 37 after seeding, we recorded baseline activity for 40 minutes those with test and control compounds and recorded for another 40 minutes. In total, we apply 12 compounds at 7 different concentrations in six replicates, so that each replicate was measured on a different MEA plate. This included positive and negative as well as solvent only controls, the advantage of this layout is that it allowsus to test robustness of the system across multiple plates and consecutive experiments.

As you can see from the depicted raster plot visualization, on the left, as well as from the plotted parameters on the right, negative compound controls show no significant effects on neuronal firing, bursting events, and network activity when compared to solvent or zero concentration. Whereas, the cytotoxic chemical tributyltin impairs all levels of neuronal activity in a dose-dependent manner. However, the application of compounds with GABA a receptor antagonistic function demonstrated an overall increase in neuronal firing with the specific changes in bursting and network activity structures. This produced long coordinated bursts at increased frequencies, indicating an organizing effect on network activity. Pyrethoids on the other hand showed an increase in neuronal firing, evidence by significant changes in spike and burst in activity at particular concentrations, but an overall and virtually complete disruption of the synchronized network activity in a dose-dependent fashion.

After changes of the neuronal activity were recorded we use the very same cultures on the MEA plates to determine the effects on cell survival. For this purpose, we applied celltiter blue staining cells and lactate dehydrogenase release assays to analyze cell viability following protocols established in the Shafer lab. Despite the drastic effects on neuronal activity seen in both GABAA blocker and pyrethroid dosing cell viability was not affected even at high compound concentrations. In contrast, the positive cytotoxic controlled tributyltin showed a dose-dependent decrease in cell survival. The system, therefore, represents a powerful tool to identify specific neuroactive effects in human neurons.

To summarize in this presentation we showed that NeuCyte's SynFire neurons serve as a defined in vitro system to study electrophysiology in a pure human cell context. The capacity of SynFire neurons to form highly synchronized network activity combined with Axion's MEA technology provides a useful platform for versatile studies of neuronal function. With regard to neural toxicity testing, we show that human neural networks respond to different chemical compounds by altering activity and organization, specifically GABAA receptor antagonists increased the spiking and natural activity in an organizing manner, in contrast, pyrethroids acting on sodium channels had a disruptive effect on neuronal networks furthermore the system can be applied to dissect neuro active from cytotoxic effects as demonstrated by the environmental neurotoxic and tributyltin.

Finally, I'm going to finish up by presenting some preliminary data that showcases the power of these phenotypic assays using the data set that we produce with the Schaefer lab. We began to treat the data analysis as a big data problem. Using a combination of AI, machine learning, and custom clustering algorithms we were able to extract the most relevant modulated features and use these to classify the compounds based on the network activity changes recorded on the MEA system. This has great potential for classifying unknown compounds by their modes of action and for disease modeling by identification of disrupted pathways and target identification.

With that said we want to thank the NeuCyteteam, the Sudhof and Wernig labs at Stanford, Tim Schafer and his team at the EPA, and InfiniteBio for the data clustering studies.

And, that is the conclusion for today's coffee break webinar if you have any questions you would like to ask regarding the research presented or if you are interested in presenting your own research with microelectrode array technology please forward them to for questions submitted for Dr. Saavedra she will be in touch with you shortly.

Thank you for joining in on today's coffee break webinar and we look forward to seeing you again.

Screening for environmental toxins with a human brain in a dish model Lorena Saavedra