Maturation and Drug Responses of hiPSC-Derived Cortical Neuronal Networks

The functional network of human-induced pluripotent stem cell (hiPSC)-derived neurons is a potentially effective in vitro model for assessing drug responses and disease mechanisms.

Nevertheless, the culture time needed for the complete functional maturation of individual neurons and networks is ambiguous. The progress of spontaneous electrophysiological activity and pharmacological responses was studied for more than 1 year in culture using multielectrode arrays (MEAs). It took 20–30 weeks for the full maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists.

During this phase, neural networks also exhibited epileptic-type synchronized burst firing (SBF) in response to SBF suppression and pro-convulsants using clinical anti-epilepsy drugs. The outcomes demonstrate the viability of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for drug screening and mechanistic analyses. However, developmental variations in pharmacological and electrophysiological properties signify the need for the international standardization of culture and evaluation practices.

Material and Methods

Human iPSC-Derived Cerebral Cortical Neurons (Axol Bioscience Inc.)

The astrocyte co-culture technique was employed to carry out long-term culture of hiPSC-derived neurons for more than 300 days.

Pyramidal neuron-like morphology and synaptogenesis at 300 DIV. (A) Scale bars = 50 μm. Green: neuronal marker β-tubulin III. Red: presynaptic marker (synaptophysin). Blue: nuclear marker (Hoechst 33258) revealing underlying astrocytes. Synapses were formed around the soma and thick dendrites. (B) Formation of pre- and postsynaptic structures at 300 DIV. Blue: nuclear marker Hoechst 33258. Green: neuronal marker β-tubulin III. Red: presynaptic marker synaptophysin. Yellow: postsynaptic marker PSD-95.

Figure 1. Pyramidal neuron-like morphology and synaptogenesis at 300 DIV. (A) Scale bars = 50 μm. Green: neuronal marker β-tubulin III. Red: presynaptic marker (synaptophysin). Blue: nuclear marker (Hoechst 33258) revealing underlying astrocytes. Synapses were formed around the soma and thick dendrites. (B) Formation of pre- and postsynaptic structures at 300 DIV. Blue: nuclear marker Hoechst 33258. Green: neuronal marker β-tubulin III. Red: presynaptic marker synaptophysin. Yellow: postsynaptic marker PSD-95.

Multi-Electrodearray System (Alpha MED Scientific Inc.)

A planar MEA measurement system (Alpha MED Scientific Inc., Japan) was employed to assess the long-term electrophysiological characteristics and drug effects of hiPSC-derived neurons. The MEA chips include 64 electrodes (MED-P515A) with a high S/N ratio and low impedance. MATLAB and Mobius software (Alpha MED Scientific Inc.) were used to carry out Spike analyses.

Result 1: Development of Spontaneous Firing During Long-Term Culture

(A) Changes in spontaneous firing pattern in the same long-term culture at 7, 14, 29, and 34 weeks in vitro (WIV). (a) Typical spontaneous firing patterns. (b) Raster plots of the array-wide spike detection rate (AWSDR, spikes/second). Bin size is 1 ms. (c) Corresponding raster plots for all 64 electrodes over 5 minutes. (B) Electrode grids color-coded to indicate mean spontaneous firing frequency from same culture at 2, 6, 14, 20, 26, and 34 WIV. Red indicates electrodes with higher firing frequencies. Scale bar in Hz (maximum: 28 Hz). (C) Time course of the average firing frequency per channel from 2 to 34 WIV. Firing frequency (±standard deviation) was calculated as the average of all 64 electrodes from each of the three MEA dishes. (D) Development of spontaneous synchronized burst firings (SBFs) during long-term culture. The number of SBFs per minute (average for 15 minutes) from 13 to 34 WIV.

(A) Changes in spontaneous firing pattern in the same long-term culture at 7, 14, 29, and 34 weeks in vitro (WIV). (a) Typical spontaneous firing patterns. (b) Raster plots of the array-wide spike detection rate (AWSDR, spikes/second). Bin size is 1 ms. (c) Corresponding raster plots for all 64 electrodes over 5 minutes. (B) Electrode grids color-coded to indicate mean spontaneous firing frequency from same culture at 2, 6, 14, 20, 26, and 34 WIV. Red indicates electrodes with higher firing frequencies. Scale bar in Hz (maximum: 28 Hz). (C) Time course of the average firing frequency per channel from 2 to 34 WIV. Firing frequency (±standard deviation) was calculated as the average of all 64 electrodes from each of the three MEA dishes. (D) Development of spontaneous synchronized burst firings (SBFs) during long-term culture. The number of SBFs per minute (average for 15 minutes) from 13 to 34 WIV.

(A) Changes in spontaneous firing pattern in the same long-term culture at 7, 14, 29, and 34 weeks in vitro (WIV). (a) Typical spontaneous firing patterns. (b) Raster plots of the array-wide spike detection rate (AWSDR, spikes/second). Bin size is 1 ms. (c) Corresponding raster plots for all 64 electrodes over 5 minutes. (B) Electrode grids color-coded to indicate mean spontaneous firing frequency from same culture at 2, 6, 14, 20, 26, and 34 WIV. Red indicates electrodes with higher firing frequencies. Scale bar in Hz (maximum: 28 Hz). (C) Time course of the average firing frequency per channel from 2 to 34 WIV. Firing frequency (±standard deviation) was calculated as the average of all 64 electrodes from each of the three MEA dishes. (D) Development of spontaneous synchronized burst firings (SBFs) during long-term culture. The number of SBFs per minute (average for 15 minutes) from 13 to 34 WIV.

(A) Changes in spontaneous firing pattern in the same long-term culture at 7, 14, 29, and 34 weeks in vitro (WIV). (a) Typical spontaneous firing patterns. (b) Raster plots of the array-wide spike detection rate (AWSDR, spikes/second). Bin size is 1 ms. (c) Corresponding raster plots for all 64 electrodes over 5 minutes. (B) Electrode grids color-coded to indicate mean spontaneous firing frequency from same culture at 2, 6, 14, 20, 26, and 34 WIV. Red indicates electrodes with higher firing frequencies. Scale bar in Hz (maximum: 28 Hz). (C) Time course of the average firing frequency per channel from 2 to 34 WIV. Firing frequency (±standard deviation) was calculated as the average of all 64 electrodes from each of the three MEA dishes. (D) Development of spontaneous synchronized burst firings (SBFs) during long-term culture. The number of SBFs per minute (average for 15 minutes) from 13 to 34 WIV.

Figure 2. (A) Changes in spontaneous firing pattern in the same long-term culture at 7, 14, 29, and 34 weeks in vitro (WIV). (a) Typical spontaneous firing patterns. (b) Raster plots of the array-wide spike detection rate (AWSDR, spikes/second). Bin size is 1 ms. (c) Corresponding raster plots for all 64 electrodes over 5 minutes. (B) Electrode grids color-coded to indicate mean spontaneous firing frequency from same culture at 2, 6, 14, 20, 26, and 34 WIV. Red indicates electrodes with higher firing frequencies. Scale bar in Hz (maximum: 28 Hz). (C) Time course of the average firing frequency per channel from 2 to 34 WIV. Firing frequency (±standard deviation) was calculated as the average of all 64 electrodes from each of the three MEA dishes. (D) Development of spontaneous synchronized burst firings (SBFs) during long-term culture. The number of SBFs per minute (average for 15 minutes) from 13 to 34 WIV.

There was a continuous increase in firing frequency and synchronized burst firings upon being cultured for nearly 20 weeks.

Result 2: Pharmacological Properties of Spontaneous Activity

Pharmacological properties of spontaneous firing activity. (A) Typical spontaneous firing at the same electrode in 33–36 WIV cultures before (top) and after the administration of 10-µM bicuculline, 5-µM kainic acid, 50-µM AP-5, and 50-µM CNQX. (B) Raster plots of spontaneous firing for 1 minute from all 64 electrodes before and after drug administration. (C) Total number of spikes from all 64 electrodes before (100%, baseline) and after drug administration at 10–15 and 33–36 WIV. Comparisons between 10–15 (gray) and 33–36 WIV (black) were obtained using the same cultures. (D) Changes in synchronized burst firing (SBF) due to bicuculline administration at 10–15 and 33–36 WIV. (a) Number of SBFs over the 30 minutes before (blue) and after (red) bicuculline administration at 10–15 and 33–36 WIV. (b) SBF duration and (c) number of spikes per SBF. (E) Number of SBFs in the 30 minutes before and after 5-µM kainic acid administration. (F) Change in SBFs following AP-5 and CNQX administration. (a) Typical SBF waveforms before (blue) and after the administration of AP-5 (red) or CNQX (red) at 33–36 WIV. SBFs disappeared after CNQX administration. SBFs were also completely abolished by AP-5 at 10–15 WIV but were only shorter at 33–36 WIV. (b) Change in SBF duration and (c) number of spikes per SBF following AP-5 administration. (n = 3 MEA dishes, *p < 0.05)

Pharmacological properties of spontaneous firing activity. (A) Typical spontaneous firing at the same electrode in 33–36 WIV cultures before (top) and after the administration of 10-µM bicuculline, 5-µM kainic acid, 50-µM AP-5, and 50-µM CNQX. (B) Raster plots of spontaneous firing for 1 minute from all 64 electrodes before and after drug administration. (C) Total number of spikes from all 64 electrodes before (100%, baseline) and after drug administration at 10–15 and 33–36 WIV. Comparisons between 10–15 (gray) and 33–36 WIV (black) were obtained using the same cultures. (D) Changes in synchronized burst firing (SBF) due to bicuculline administration at 10–15 and 33–36 WIV. (a) Number of SBFs over the 30 minutes before (blue) and after (red) bicuculline administration at 10–15 and 33–36 WIV. (b) SBF duration and (c) number of spikes per SBF. (E) Number of SBFs in the 30 minutes before and after 5-µM kainic acid administration. (F) Change in SBFs following AP-5 and CNQX administration. (a) Typical SBF waveforms before (blue) and after the administration of AP-5 (red) or CNQX (red) at 33–36 WIV. SBFs disappeared after CNQX administration. SBFs were also completely abolished by AP-5 at 10–15 WIV but were only shorter at 33–36 WIV. (b) Change in SBF duration and (c) number of spikes per SBF following AP-5 administration. (n = 3 MEA dishes, *p < 0.05)

Figure 3. Pharmacological properties of spontaneous firing activity. (A) Typical spontaneous firing at the same electrode in 33–36 WIV cultures before (top) and after the administration of 10 µM bicuculline, 5 µM kainic acid, 50 µM AP-5, and 50 µM CNQX. (B) Raster plots of spontaneous firing for 1 minute from all 64 electrodes before and after drug administration. (C) Total number of spikes from all 64 electrodes before (100%, baseline) and after drug administration at 10–15 and 33–36 WIV. Comparisons between 10–15 (gray) and 33–36 WIV (black) were obtained using the same cultures. (D) Changes in synchronized burst firing (SBF) due to bicuculline administration at 10–15 and 33–36 WIV. (a) Number of SBFs over the 30 minutes before (blue) and after (red) bicuculline administration at 10–15 and 33–36 WIV. (b) SBF duration and (c) number of spikes per SBF. (E) Number of SBFs in the 30 minutes before and after 5 µM kainic acid administration. (F) Change in SBFs following AP-5 and CNQX administration. (a) Typical SBF waveforms before (blue) and after the administration of AP-5 (red) or CNQX (red) at 33–36 WIV. SBFs disappeared after CNQX administration. SBFs were also completely abolished by AP-5 at 10–15 WIV but were only shorter at 33–36 WIV. (b) Change in SBF duration and (c) number of spikes per SBF following AP-5 administration. (n = 3 MEA dishes, *p < 0.05)

Spontaneous activity in 33–36 WIV cultures was more sensitive to glutamate receptor modulators and GABA.

Result 3: Pharmacological Properties of Evoked Responses

Pharmacological properties of evoked responses. (A) Typical evoked responses from each of 64 electrodes following a single test stimulus at 33–36 WIV. Red square shows stimulus site (electrode 33). (B) Typical evoked responses before (top) and after the administration of the indicated neurotransmitter receptor agonist or antagonist at 33–36 WIV. Red triangle shows stimulus time and stimulus artifacts. (C) Post-stimulus time histogram (PSTH) (Sum of 10 individual responses at 64 electrodes, bin size = 1 ms) at 33–36 WIV. Blue and red indicate before and after drug administration, respectively. (D) Ratio of the number of evoked spikes after versus before drug administration at 10–15 WIV (gray) and 33–36 WIV (black) (n = 3 MEA dishes, *p < 0.05).

Pharmacological properties of evoked responses. (A) Typical evoked responses from each of 64 electrodes following a single test stimulus at 33–36 WIV. Red square shows stimulus site (electrode 33). (B) Typical evoked responses before (top) and after the administration of the indicated neurotransmitter receptor agonist or antagonist at 33–36 WIV. Red triangle shows stimulus time and stimulus artifacts. (C) Post-stimulus time histogram (PSTH) (Sum of 10 individual responses at 64 electrodes, bin size = 1 ms) at 33–36 WIV. Blue and red indicate before and after drug administration, respectively. (D) Ratio of the number of evoked spikes after versus before drug administration at 10–15 WIV (gray) and 33–36 WIV (black) (n = 3 MEA dishes, *p < 0.05).

Figure 4. Pharmacological properties of evoked responses. (A) Typical evoked responses from each of 64 electrodes following a single test stimulus at 33–36 WIV. Red square shows stimulus site (electrode 33). (B) Typical evoked responses before (top) and after the administration of the indicated neurotransmitter receptor agonist or antagonist at 33–36 WIV. Red triangle shows stimulus time and stimulus artifacts. (C) Post-stimulus time histogram (PSTH) (Sum of 10 individual responses at 64 electrodes, bin size = 1 ms) at 33–36 WIV. Blue and red indicate before and after drug administration, respectively. (D) Ratio of the number of evoked spikes after versus before drug administration at 10–15 WIV (gray) and 33–36 WIV (black) (n = 3 MEA dishes, *p < 0.05).

Evoked responses at 33–36 WIV vividly exhibit the differences between NMDA and AMPA receptor expression and response.

Result 4: Induction of Epileptiform Activity and Effects of Anti-Epilepsy Drugs

Induction of epileptiform activity and anticonvulsant effects of anti-epilepsy drugs (AEDs). (A) Induction of epileptiform activity using pentylenetetrazole (PTZ) and the suppressive effect of phenytoin. PTZ was added at increasing concentrations (1 µM, 10 µM, 100 µM, and 1 mM). Phenytoin was then added (1 µM, 10 µM, 100 µM, and 1 mM). The raster plots at 20 WIV (139 DIV). (B) Changes in firing rate versus before (%) and number of SBFs (Yellow). (C) Effect of sodium valproate (VPA) (1 µM, 10 µM, 100 µM, 1mM, and 2 mM). The raster plots at 15 WIV (99 DIV). (D) Changes in firing rate and number of SBFs (MEA dishes, *p < 0.05).

Induction of epileptiform activity and anticonvulsant effects of anti-epilepsy drugs (AEDs). (A) Induction of epileptiform activity using pentylenetetrazole (PTZ) and the suppressive effect of phenytoin. PTZ was added at increasing concentrations (1 µM, 10 µM, 100 µM, and 1 mM). Phenytoin was then added (1 µM, 10 µM, 100 µM, and 1 mM). The raster plots at 20 WIV (139 DIV). (B) Changes in firing rate versus before (%) and number of SBFs (Yellow). (C) Effect of sodium valproate (VPA) (1 µM, 10 µM, 100 µM, 1mM, and 2 mM). The raster plots at 15 WIV (99 DIV). (D) Changes in firing rate and number of SBFs (MEA dishes, *p < 0.05).

Induction of epileptiform activity and anticonvulsant effects of anti-epilepsy drugs (AEDs). (A) Induction of epileptiform activity using pentylenetetrazole (PTZ) and the suppressive effect of phenytoin. PTZ was added at increasing concentrations (1 µM, 10 µM, 100 µM, and 1 mM). Phenytoin was then added (1 µM, 10 µM, 100 µM, and 1 mM). The raster plots at 20 WIV (139 DIV). (B) Changes in firing rate versus before (%) and number of SBFs (Yellow). (C) Effect of sodium valproate (VPA) (1 µM, 10 µM, 100 µM, 1mM, and 2 mM). The raster plots at 15 WIV (99 DIV). (D) Changes in firing rate and number of SBFs (MEA dishes, *p < 0.05).

Induction of epileptiform activity and anticonvulsant effects of anti-epilepsy drugs (AEDs). (A) Induction of epileptiform activity using pentylenetetrazole (PTZ) and the suppressive effect of phenytoin. PTZ was added at increasing concentrations (1 µM, 10 µM, 100 µM, and 1 mM). Phenytoin was then added (1 µM, 10 µM, 100 µM, and 1 mM). The raster plots at 20 WIV (139 DIV). (B) Changes in firing rate versus before (%) and number of SBFs (Yellow). (C) Effect of sodium valproate (VPA) (1 µM, 10 µM, 100 µM, 1mM, and 2 mM). The raster plots at 15 WIV (99 DIV). (D) Changes in firing rate and number of SBFs (MEA dishes, *p < 0.05).

Figure 5. Induction of epileptiform activity and anticonvulsant effects of anti-epilepsy drugs (AEDs). (A) Induction of epileptiform activity using pentylenetetrazole (PTZ) and the suppressive effect of phenytoin. PTZ was added at increasing concentrations (1 µM, 10 µM, 100 µM, and 1 mM). Phenytoin was then added (1 µM, 10 µM, 100 µM, and 1 mM). The raster plots at 20 WIV (139 DIV). (B) Changes in firing rate versus before (%) and number of SBFs (Yellow). (C) Effect of sodium valproate (VPA) (1 µM, 10 µM, 100 µM, 1mM, and 2 mM). The raster plots at 15 WIV (99 DIV). (D) Changes in firing rate and number of SBFs (MEA dishes, *p < 0.05).

The induction of epileptiform activity by PTZ and suppressive impacts by clinical AEDs (phenytoin and VPA) were also detected.

Conclusion

To conclude, the pharmacological and electrophysiological properties of cultured hiPSC-derived cortical neuronal networks were studied and it was identified that functional maturation needs at least 20–30 weeks. However, long-term culture of hiPSC-derived neuronal neurons on MEAs was established to be helpful for neurotoxicological and neuropharmacological assays. Furthermore, their outcomes offer a vital indication for the international standardization of assessment procedures using in vitro human neurons.

Reference

  • Odawara A., Katoh H., Matsuda N., Suzuki I. Sci Rep. 2016, 6:26181.

About AXOL Biosciences

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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.

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Last updated: Oct 28, 2019 at 10:54 AM

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