The functional network of human-induced pluripotent stem cell (hiPSC)-derived neurons is a highly potent in vitro model for assessing drug responses and disease mechanisms.
Yet, the culture time needed for the complete functional maturation of individual neurons and networks is unpredictable. The progress of spontaneous electrophysiological activity and pharmacological responses was examined for more than one year in culture using multi-electrode arrays (MEAs). About 20–30 weeks were needed 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 epileptiform synchronized burst firing (SBF) as a result of pro-convulsants and SBF suppression using clinical anti-epilepsy drugs.
The outcomes exhibit the feasibility 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 represent the need for the international standardization of culture and evaluation procedures.
Material and Methods
Human iPSC-Derived Cerebral Cortical Neurons (Axol Bioscience Inc.)(1)
- Using astrocyte co-culture method, (2) long-term culture of hiPSC-derived neurons were carried out for more than 300 days.
Figure 1. Pyramidal neuron-like morphology and synaptogenesis at 300 DIV. (A) Scale bars = 50 μm.
Multi-Electrode Array System (Alpha MED Scientific Inc.)
- The long-term electrophysiological characteristics and drug effects of hiPSC-derived neurons were assessed using a planar MEA measurement system (Alpha MED Scientific Inc., Japan). The MEA chips consist of 64 electrodes (MED-P515A) with high S/N ratio and low impedance.
- Spike analyses were carried out using MATLAB and Mobius software (Alpha MED Scientific).
Result 1: Development of Spontaneous Firing During Long-Term Culture
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 up to 20 weeks.
Result 2: Pharmacological Properties of Spontaneous Activity
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).
Glutamatergic receptors are more functional at 33–36 WIV than at 10–15 WIV.
Result 3: Pharmacological Properties of Evoked Responses
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). (E) The ratio of evoked burst duration after versus before drug administration (n = 3 MEA dishes, *p < 0.05).
The differences between AMPA and NMDA receptor expression and response are evidently shown by evoked responses at 33–36 WIV.
Result 4: Induction of Epileptiform Activity and Effects of Anti-Epilepsy Drugs
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, 1 mM, and 2 mM). The raster plots at 15 WIV (99 DIV). (D) Changes in firing rate and number of SBFs.
The induction of epileptiform activity by PTZ and suppressive effects by clinical AEDs are detected.
To sum up, the electrophysiological and pharmacological properties of cultured hiPSC-derived cortical neuronal networks were studied and it was discovered that at least 20–30 weeks were needed for functional maturation. Nevertheless, long-term culture of hiPSC-derived neuronal neurons on MEAs was shown to be valuable for neurotoxicological and neuropharmacological assays. In addition, the outcomes offer a key indication for the international standardization of assessment procedures using in vitro human neurons.
(1) Shi Y, Kirwan P, Livesey FJ. Nature Protocol 2012, 7: 1836-1846.
(2) Odawara A, Katoh H, Matsuda N, Suzuki I. Biochem Biophys Res Commun 2016, 469(4): 856-62.
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.
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