Electrophysiological Drug Testing using hiPSC-Derived Neurons

Neurons derived from human-induced pluripotent stem cells (hiPSCs) can be effectively used for cell-based therapy and drug discovery.

This article demonstrates how a multi-electrode array (MEA) system is used for investigating the functional properties of hiPSC-derived neurons on their drug responsiveness and long-term spontaneous activity over a culture period of 300 days.

Following 70 days of culture, a synchronous burst firing activity was observed owing to synapse transmission inside the neuronal networks. The firing rate in spontaneous firings and electrically evoked responses underwent considerable changes when the synapse agonist and antagonists—bicuculline, kainic acid, AP5, and CNQX—were added.

It was also shown that epilepsy phenomenon was triggered upon administrating pentylenetetrazole (PTZ) and was suppressed upon injecting phenytoin and sodium valproate (VPA), an anti-epilepsy drug.

PTZ 100 μM triggered high-frequency synchronized bursts, but these were slowly reduced by increasing the dose of an anti-epilepsy drug. The bursts subsequently disappeared over phenytoin 100 μM or VPA 1 mM, respectively.

These outcomes show that using an MEA system for long-term electrophysiological measurements in hiPSC-derived neurons may prove useful for drug screening applications.

Material and Methods

(A) Long-Term Electrophysiological Measurement

(B) Human iPSC-Derived Neurons

Maturation hiPSC-derived neurons after 300 days of culture

  • A planar MEA measurement system (Alpha Med Scientific, Japan) was used to assess the drug effect and long-term electrophysiological feature of the hiPSC-derived neurons. The MEA chips constituted 64 electrodes (MED-P515A) with high S/N ratio and low impedance.
  • hiPSC-derived neurons (Cerebral Cortical Neurons; Axol Bioscience Inc., UK) (1) were cultured on MEA chips. Astrocyte co-culture method was used to perform a long-term culture of hiPSC-derived neurons.(2)
  • Mobius software (Alpha Med Scientific) and MATLAB were used to perform firing analyses.

Result 1—Time Course of Spontaneous Firings

Time course of spontaneous firing over 200 days of culture in different culture conditions. (A) The waveforms and laster plot at 64 electrodes. (B) Number of total spontaneous spikes detected vs. the time course. (C) Grids showing the 64 electrodes where colored electrodes detected signals. Electrodes that detected a higher firing frequency are shown in red.

Figure 1. Time course of spontaneous firing over 200 days of culture in different culture conditions. (A) The waveforms and laster plot at 64 electrodes. (B) Number of total spontaneous spikes detected vs. the time course. (C) Grids showing the 64 electrodes where colored electrodes detected signals. Electrodes that detected a higher firing frequency are shown in red.

  • Synchronized burst firings were seen over a culture time of 70 days
  • Synchronized burst firings and firing rate increased up to approximately 200 days

Result 2—Drug Effect in Spontaneous Firings

Effects of drugs in spontaneous firings at 100 culture days in vitro and 240 culture days in vitro (DIV) in the same samples. (A) The waveforms represent typical changes in spontaneous firings after Bicuculline 10 µM, Kainic acid 5 µM, CNQX 50 µM, and AP5 50 µM administration at 240 DIV. (B) Laster plot at 64 electrodes after drug administration. (C) Percentage of the number of total spikes vs. before (n=3 MEA dishes, *P<0.05). (D) Fast Fourier Transform (FFT) of spontaneous firing after different drug administration. (E) (a) Synchronized burst counts of before and after different drug administration at 100 DIV and 240 DIV (*P<0.05). (b) Duration of synchronized burst before and after Bicuculline and Kainic acid administration (*P<0.05).

Effects of drugs in spontaneous firings at 100 culture days in vitro and 240 culture days in vitro (DIV) in the same samples. (A) The waveforms represent typical changes in spontaneous firings after Bicuculline 10 µM, Kainic acid 5 µM, CNQX 50 µM, and AP5 50 µM administration at 240 DIV. (B) Laster plot at 64 electrodes after drug administration. (C) Percentage of the number of total spikes vs. before (n=3 MEA dishes, *P<0.05). (D) Fast Fourier Transform (FFT) of spontaneous firing after different drug administration. (E) (a) Synchronized burst counts of before and after different drug administration at 100 DIV and 240 DIV (*P<0.05). (b) Duration of synchronized burst before and after Bicuculline and Kainic acid administration (*P<0.05).

Figure 2. Effects of drugs in spontaneous firings at 100 culture days in vitro and 240 culture days in vitro (DIV) in the same samples. (A) The waveforms represent typical changes in spontaneous firings after Bicuculline 10 µM, Kainic acid 5 µM, CNQX 50 µM, and AP5 50 µM administration at 240 DIV. (B) Laster plot at 64 electrodes after drug administration. (C) Percentage of the number of total spikes vs. before (n=3 MEA dishes, *P<0.05). (D) Fast Fourier Transform (FFT) of spontaneous firing after different drug administration. (E) (a) Synchronized burst counts of before and after different drug administration at 100 DIV and 240 DIV (*P<0.05). (b) Duration of synchronized burst before and after Bicuculline and Kainic acid administration (*P<0.05).

  • Kainic acid increased the number of synchronized burst firings, and bicuculline increased the duration of synchronized burst firings. However, burst firings disappeared after the administration of AP5 and CNQX.
  • Functional ion channel response at 240 DIV was found to be better than the responses at 100 DIV.
  • High-frequency firings were induced by bicuculline. Firings frequency was different based on the type of synaptic’s drug.

Result 3—Drug effect in Evoked Responses

Effects of drugs in electrically evoked response. (A) The waveforms represent typical changes in the evoked responses after drug administration at 240 culture days in vitro (DIV). (B) Peri-stimulus time histogram (PSTH) of 120 trace data at an electrode. (C) Percentage of duration in a burst and number of spikes in a burst before and after drug administration at 100 DIV and 240 DIV (*P < 0.01).

Figure 3. Effects of drugs in electrically evoked response. (A) The waveforms represent typical changes in the evoked responses after drug administration at 240 culture days in vitro (DIV). (B) Peri-stimulus time histogram (PSTH) of 120 trace data at an electrode. (C) Percentage of duration in a burst and number of spikes in a burst before and after drug administration at 100 DIV and 240 DIV (*P < 0.01).

  • Electrical stimulation revealed the effects of synaptic’s drug in evoked responses
  • The number and duration of spikes in a burst were different based on the type of synaptic’s drug
  • When compared to the evoked responses at 100 DIV, the change in the triggered responses was found to be significant
  • AP5 considerably reduced the duration of the burst. This outcome indicates the functioning NMDA receptors

Result 4—Epilepsy Phenomenon and Drug Effects

Evoked epilepsy phenomenon and the effects of anti-epilepsy drugs. (A) Laster plot in PTZ and VPA administration. (B) Laster plot in PTZ and Phenytoin administration. (C) Changes in firing rate and burst count.

Figure 4. Evoked epilepsy phenomenon and the effects of anti-epilepsy drugs. (A) Laster plot in PTZ and VPA administration. (B) Laster plot in PTZ and Phenytoin administration. (C) Changes in firing rate and burst count.

Administration of pentylenetetrazole (PTZ) evoked epilepsy phenomenon, but this was suppressed by the anti-epilepsy drug—phenytoin and sodium valproate (VPA).

Conclusion

  • The hiPSC-derived neurons enabled a long time culture for over 300 days and also allowed the functional maturation utilizing co-culture with astrocyte
  • Long-term electrophysiological features and the effects of synaptic’s drugs in cultured hiPSC-derived neurons were successfully detected
  • Compared to 100 days of culture, hiPS-derived neurons were observed to be more functional at 240 days of culture
  • Triggered responses by electrical stimulation are valuable for assessing the drugs’ effects
  • Effects of anti-epilepsy drug and detection of epilepsy phenomenon indicate a human epilepsy model for drug screening
  • The use of multi-electrode arrays for long-term electrophysiological measurement enabled toxicological assay and drug screening

Reference

  1. Shi Y, Kirwan P, Livesey FJ. Nature Protocols 2012, 7: 1836-1846.
  2. (2) Odawara A, Sitoh Y. Gotoh M. Suzuki I. Biochemical and Biophysical Research Communications 2014, 443: 1176-1181

About AXOL Biosciences

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Last updated: Nov 4, 2019 at 8:22 AM

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