Evaluating Convulsive Activity in hiPSC-Derived Neurons and Astrocytes using a Micro-Electrode Array

In vitro assays utilizing human induced pluripotent stem cell (hiPSC)-derived neurons provide a persuasive alternative to animal models for safety screening and drug discovery. A possible problem when utilizing hiPSC-derived neuron culture assays is understanding the degree in which in vitro data predicts in vivo effects of investigational drug compounds.

To solve this challenge between in vitro and in vivo assays, a high-sensitivity micro-electrode array (MEA) was employed to verify whether human neurons in vitro exhibit activity that can be extrapolated to activity that is exhibited in vivo.

In particular, a low-noise and high-sensitivity MEA, the MED64 Presto, was utilized to prove that hiPSC-derived neurons exhibit slow-wave oscillations that are similar to patterns of brain activity as measured by human electroencephalogram (EEG) and ECoG.

MEAs are best adapted to show possible neurotoxic effects because of the ability of MEAs to quantify seizure-like convulsant activity in hiPSC-derived neuron cultures. Convulsant activity is observed by quantifying synchronized burst activity and spiking.

Similarly, in vivo EEG oscillations illustrate the synchronized activity over a network of brain neurons. The MED64 Presto is uniquely manufactured for low-noise and high-sensitivity. The low noise of the MED64 Presto allows for raw data logging with reduced filtering so that the slow-wave oscillations between 1Hz and 250 Hz can be analyzed and extracted.

This article outlines the power of the MED64 Presto in predicting convulsion toxicity utilizing hiPSC-derived neurons and the remarkable similarity between the frequency of in vitro and in vivo slow-wave oscillations.

Materials and Methods

Slow Wave Oscillations

To analyze slow-wave oscillations, Axol™ Human iPSC-Derived Neural Stem Cells and Mature Astrocyte were co-cultured onto a 24-well MED64 Presto plate (Figure 1C). Raw data was recorded at an acquisition bandwidth of 0.1Hz – 5 kHz.

Slow wave oscillations of synchronized burst firing (SBFs) were investigated from local field potentials (LFPs) at a bandwidth of between 1Hz and 250 Hz, frequencies commonly associated with in vivo ECoG and EEG data (Figure 1B). A high sensitivity MEA is necessary so that the slow-wave oscillations are not covered by exogenous noise, for example 60Hz noise that would permeate MEAs that are less sensitive (Figure 1E).

A) EEG electrodes placed on the scalp of a human or animal can measure local field potentials (B) from synchronized activity in the brain. (C) hiPSC-derived neuron and astrocyte co-culture are shown cultured on a grid of electrode. (D) Slow-wave activity measured at each electrode with the MED64 Presto. (E) The MED64 Presto that can record field potentials from cells cultured on a 16-electrode grid per well.

Figure 1: A) EEG electrodes placed on the scalp of a human or animal can measure local field potentials (B) from synchronized activity in the brain. (C) hiPSC-derived neuron and astrocyte co-culture are shown cultured on a grid of electrode. (D) Slow-wave activity measured at each electrode with the MED64 Presto. (E) The MED64 Presto that can record field potentials from cells cultured on a 16-electrode grid per well.

Frequency Analysis

Frequencies over 250 Hz were taken from the LFP raw data by a band pass finite impulse response (FIR) filter from 0.1 to 250 Hz utilizing the Signal Processing Toolbox in MatLab (Figure 2). The filter was applied forward and in reverse to remove any phase distortions.

Wavelet measurements of LFP were carried out employing a customized program in MatLab (using function cwt () in the “Wavelet Toolbox”).

Validation Data

Decomposition into Frequency Components

Burst activity is detected from the local field potential measured at each electrode. Wavelet of the SBF is performed on the raw data logged to decompose SBF into frequency components. The power of the frequency component is quantified within frequency bands analogous to EEG.

Figure 2: Burst activity is detected from the local field potential measured at each electrode. Wavelet of the SBF is performed on the raw data logged to decompose SBF into frequency components. The power of the frequency component is quantified within frequency bands analogous to EEG.

Pharmacologically Induced Activity

To understand the differences and similarities between in vivo brain activity and in vitro human iPSC-derived neurons, the amplitude of the wavelet power spectrum from each LFP was recorded in response to greater concentrations 4-Aminopyridine (4-AP), a K+ channel blocker that produces seizure-like activity.

The amplitude of the wavelet power spectrum was greater in response to the 4-AP in the β and γ wave components. These outcomes are incredibly similar to in vivo epileptiform EEG activity (Sitges et al. 2016) showing that in vitro high sensitivity MEA assays provide a persuasive alternative to animal models for safety screening and drug discovery.

The strength of the β and γ wave components are also similar to what has been observed in human EEG epileptiform activity (Gollwitzer et al. 2016) showing that hiPSC derived neurons in vitro may be a predictive model for in vivo results of investigational drug compounds. As a whole, these results reveal the potential of hiPSC assay with a high-sensitivity MEA for neurotoxicology screening.

Functional Characterization

The MED64 Presto high-sensitivity high-throughput MEA paired with Axol™ Human iPSC-Derived Neural Stem Cells and Mature Astrocyte have been verified as the best set of tools for assessing epileptiform activity in vitro.

This article proves that it is possible to calculate slow-wave frequency components utilizing the MED64 Presto, the slow-wave frequency components extrapolate to in vivo responses, and are similar to human in vivo EEG data.

Moreover, this article shows that the MED64 Presto can be utilized to screen for possible neurotoxic events of investigational drug compounds. For example, Figure 3 demonstrates a strong drug effect of 4-AP as determined by slow-wave frequency oscillations where the power spectrum changed in response to greater concentrations of 4-AP.

Seizurogenic activity is a possible outcome of investigational drug compounds and the ability to analyze seizure-like activity in vitro is critical for pre-clinical safety screening and drug development.

A) Local Field Potential and wavelet analysis in response to 0 to 10 μM 4-AP administration. The x-axis of the spectrogram represents time and the Y-axis the frequency component. Power spectrum shifted to the beginning portion of the burst with increased concentration of 4-AP (red circle). (B) The power difference was strongest in the β and γ wave frequency components, similar to what has been reported on in vivo EEG data.

Figure 3: A) Local Field Potential and wavelet analysis in response to 0 to 10 μM 4-AP administration. The x-axis of the spectrogram represents time and the Y-axis the frequency component. Power spectrum shifted to the beginning portion of the burst with increased concentration of 4-AP (red circle). (B) The power difference was strongest in the β and γ wave frequency components, similar to what has been reported on in vivo EEG data.

Conclusion

The assessment of antiepileptic drugs utilizing human iPSC-derived neurons with the MED64 Presto is a beneficial system for adverse event detection and drug discovery. The MED64 is characterized by its low-noise and high-sensitivity which makes it the best platform for analyzing the positive effects of antiepileptic drugs, along with the mechanisms of action of antiepileptic drugs and possible adverse events.

The MED64 Presto has the greatest sensitivity and highest throughput MEA on the market. Its amplifier and MEA Plate are engineered to optimize sensitivity. A wide acquisition bandwidth allows for the ability to record the diversity of responses from cultured cells.

Although, it must be noted that some MEAs compromise signal-to-noise which can cause the loss of important signals. MED64 Presto’s high-sensitivity gives superior signal-to-noise even with a broad acquisition bandwidth. MED64 Presto can measure a wide spectrum of possible action potentials from neuron culture, creating more reliable and reproducible data.

Being able to extract slow-wave oscillations is dependent on having a low-noise platform that can log raw data. Raw data logging with the least amount of filtering is a unique feature of the MED64 Presto and is the critical feature that allows analysis of several frequency components. This makes the MED64 Presto perfect for estimating the in vivo effects of investigational drug compounds.

It is critical to show an association between in vitro and in vivo convulsive activity in order to develop a convulsion prediction system in vitro. To display the similarities between in vitro neuron firing and in vivo brain activity, frequency bands under 250 Hz (similar to EEG frequency range) were measured utilizing the MED64 Presto.

A dose-dependent response was measured in the β and γ wave frequency components responding to 4-AP administration, the same as what has been observed from EEG recordings in vivo. As such, this article shows the capabilities of the MED64 Presto to forecast in vivo effects.

References and Further Reading

  • llwitzer, S. et al. Visual and semiautomated evaluation of epileptogenicity in focal cortical dysplasias - An intracranial EEG study. Epilepsy & behavior: E&B 58, 69–75, (2016).
  • Odawara A., Matsuda, N., Ishibashi, Y., Yokoi, R., Suzuki, I. Toxicological evaluation of convulsant and anticonvulsant drugs in human induced pluripotent stem cell-derived cortical neuronal networks using a MEA system, Scientific Reports 8:10416 (2018).
  • Sitges, M., Aldana, B. I. & Reed, R. C. Effect of the Antidepressant Sertraline, the Novel Anti-seizure Drug Vinpocetine and Several Conventional Antiepileptic Drugs on the Epileptiform EEG Activity Induced by 4-Aminopyridine. Neurochemical research 41, (2016).
  • References Copyright: 2018 Alpha MED Scientific Inc. All rights reserved. Alpha MED Scientific will not guarantee that the same results will be obtained using the MED64 Presto. APN180901_Ver1.2
  • All Data: provided by Ikruo Suzuki, PhD, Tohoku Institute of Technology

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Nov 18, 2019 at 8:08 AM

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