Electrophysiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks over 1 years culture
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1
Tohoku Institute of Technology, Department of Electronics, Japan
Motivation
Human induced pluripotent stem cell (hiPSC)-derived neurons may be effectively used for drug discovery and toxicity testing. However, the immaturity of cultured human iPSC-derived neurons and the lack of established functional evaluation methods are problematic. The purpose of this study was to evaluate the physiological maturation and drug responses depending on culture days using multi-electrode array (MEA) system, and to detect the epilepsy pehnomena and the effects of ani epilepsy drugs as an application example.
Material and Methods
Human induced PSC-derived cerebral cortical neurons (hyCCNs; ax0026F, Axol Bioscience Inc., UK) [1] were cultured at 1.0 ~ 106 cells/cm2 on 64-channel MEA chips (MED-P515A; Alpha Med Scientific) coated with Axol Sure Bond Coating Solution (Axol Sure Bond Coating solusion; ax0041, Axol Bioscience Inc.) at 37‹C in a 5% CO2/95% air atmosphere. Spontaneous and evoked extracellular field potentials were acquired at 37‹C under a 5% CO2 atmosphere using a 64-channel MEA system (MED64-Basic; Alpha Med Scientific) at a sampling rate of 20 kHz/channel. Signals were low-pass filtered at 100 Hz and stored on a personal computer. Spontaneous firing was recorded every week for over 600 days. To investigate pharmacological effects depending culture days, we administered 10 ƒÊM bicuculline, 5 ƒÊM kainic acid, 50 ƒÊM CNQX, and 50 ƒÊM AP-5 at 10-15 and 33-36 weeks culture respectively. To detect the epilepsy pehnomena and the effects of ani-epilepsy drugs (AEDs), we administerd pentylentetrazole (PTZ) and typical AEDs (phenytoin and sodium valproate (VPA).
Results
We have succeeded in detection of spontaneous firings over 600 days and the future of time course. Syncronized burst firings (SBFs), in which generated by chemical synapse transmission, were observed over 10-13 weeks. As the culture days has passed, the number of occurrences of SBFs were increased up to approximately 30 weeks. Both spontaneous firings and evoked responses, we detected the effects of typical synaptic-related drugs at 33-36 weeks compaird to 10-15 weeks culture, indicative of greater functional maturation. Moreover, we also detected the induction of epileptic seizure by PTZ, in which high frequency SBFs were generated by 0.1-1mM PTZ administration, and the effects of AEDs [2].
Discussion
Synchronized spike bursts are critical for information transfer within the cortex and are indicators of functional maturation. Such SBFs were first observed at 10?13 weeks and increased in frequency up to approximately 30 weeks. In rat cortical neurons at 1/4 cell density, synchronized firing attributable to chemical synaptic transmission occurred after only 1 week in culture, suggesting that hiPSC-derived cortical neurons require much longer to achieve functional maturation in vitro. Nonetheless, this extra time is reasonable considering the advantages for translational research presented by human neurons.
These spontaneous and evoked responses also showed progressive changes in pharmacological response. In particular, the detection of fully functional NMDA responses is important because the NMDA receptor mediates several forms of neuroplasticity related to learning. Moreover, NMDA overactivity is a key pathogenic event in numerous acute and chronic neurodegenerative diseases.
We also suggest that the human iPSC-derived neuron culture is a useful model system to investigate the effects of common AEDs and an alternative to animal experiments for drug screening. In addition, our assay can be adapted to iPSC-derived neurons from epilepsy patients.
Conclusion
We found that functional maturation in hiPSC-derived coritical neurons need the long-term culture over 20-30 weeks. Evaluation of drug responses is suitable to the culture sample over 20- 30 weeks. We have proved the MEA coupled hiPSC-derived neuronal neurons is useful to neuropharmacological/neurotoxicological assays for drug screening by detecting the drug effects both spontaneous firings and evoked responses. Our results also provide an important indication for the international standardization of evaluation procedures using in vitro human central neurons [2].
Acknowledgements
We thank Axol Bioscience and Alpha Med Scientific Company for supporting this research. This study was supported by JSPS KAKENHI Grant Number 26560247, 14J11194.
References
[1] Shi, Y., Kirwan, P. & Livesey, F. J. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc 7, 1836-1846, doi:10.1038/nprot.2012.116 (2012).
[2] Odawara A, Katoh H, Matsuda N, Suzuki I. Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture. Sci Rep. 6, 26181 (2016) doi:10.1038/srep26181
Figure Legend
(A) Development of spontaneous firing during a long-term culture of hiPSC-derived cortical neurons. Changes in spontaneous firing pattern in the same long-term culture at 7, 14, and 29 weeks. Raster plots for all 64 electrodes over 5 min.(B) Pharmacological properties of evoked responses. (a)Typical evoked responses before and after the administration of the indicated neurotransmitter receptor agonist or antagonist at 33?36 weeks in vitro. (b)Post-stimulus time histogram (PSTH) (Sum of 10 individual responses at 64 electrodes, bin size = 1 ms). Blue and red indicate before and after drug administration, respectively. (C) Induction of epileptiform activity and anticonvulsant effects of anti-epilepsy drugs. Effects on network activity are clearly revealed by the raster plots (64 channels for 5 min). The appearance of black vertical lines indicates SBFs. Created based upon data from [2].
Keywords:
Epilepsy,
Long-term culture,
hiPSC-derived cerebral cortical,
Pharmacological responses
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
oral
Topic:
MEA Meeting 2016
Citation:
Suzuki
I,
Odawara
A,
Katoh
H and
Matsuda
N
(2016). Electrophysiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks over 1 years culture.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00067
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Ikuro Suzuki, Tohoku Institute of Technology, Department of Electronics, Sendai, Japan, i-suzuki@tohtech.ac.jp