Comparing drug-induced seizure-liability in human iPSC-derived and primary mouse neurons grown on micro-electrode arrays
NeuroProof GmbH, Germany
NeuroProof GmbH, Germany
The high attrition rate in pharmaceutical compounds is partly caused by their toxic effects on the central nervous system. Seizure side effects cause phases of hyper synchronous excitation which often lead to convulsive motor phenotypes. Until now, seizure liability is tested in complex, expensive and low throughput ex vivo rat hippocampal brain slices. Several studies have shown that in vitro rodent cortex cultures grown on micro-electrode arrays can be used for recording seizure-like activity. These cortex cultures retain many features with the tissues from which they are obtained, including cell phenotypes, receptor and ion channel composition, intrinsic electrical membrane properties, synaptic development and plasticity. Thus, calibrating in vitro systems with known seizure-related compounds is currently assessed as an alternative to slice assays. However, to improve predictability, sensitivity and specificity human induced pluripotent stem cell-derived (hiPSC) neurons are relevant to transfer the current models into the human background. Nonetheless, mouse in vitro cultures are still required to calibrate novel human-derived cell culture models. The aim of this study was to compare toxin-induced functional phenotypes in mouse and hiPSC-derived neuronal cultures.
Material and Methods
Primary cell cultures. Embryonic frontal cortex tissue was dissected at embryonic day 15 and cultured for 28 days on microelectrode arrays (MEAs) (Center for Network NeuroScience, University of North Texas, Denton, TX) with 2x32 passive electrodes as previously described (Hammer, Bader et al. 2015).
Human iPSC-derived neuronal cultures: Frozen human iPSC-derived neurons (Dopa.4U, Axiogenesis, Germany) were thawed, plated on 12-well MEAs (Axion Biosystems, USA, 64 electrodes per well) and cultured according to the manufacturerÕs instructions.
Multichannel recordings and data analysis. Extracellular recordings were performed using MEA workstation acquisition systems from Plexon and Axion Biosystems (Maestro system) for the two MEA systems, respectively. Both systems allow temperature control of 37¡C and stable pH of 7.4 (5-10% CO2), thus enabling stable recording and cumulative concentration-response experiments. On both, mouse and human cell cultures compounds were added with cumulatively increasing concentrations (maximum assay concentration of DMSO: 0.1%). Each concentration episode was recorded for 60 minutes whereby a stable activity phase of 30 min was analyzed. Spike train acquisition was performed using the programs NeuroEXplorer (Plexon Inc., Dallas, TX) and Axis (Axion Biosystems, USA). Multivariate analysis was performed using proprietary tool NPWaveX for description of the activity changes in four defined categories: overall activity, burst structure, oscillatory behavior and synchronicity (see Hammer, Bader et al. 2015). Abbreviations: ISI = inter spike interval in burst, CVnet = coefficient of variation in network, SD = standard deviation within recorded 30 min time frame. Number of repeats: 3-6. Statistical analysis: unpaired t-test vs. native with *p²0.05, **p²0.01, ***p²0.001.
In order to compare compound effects between mouse cortex cultures and human iPSC-derived neurons, comparable activity patterns are required as the patterns reflect the cellular complexity and connectivity within the cultures. After optimizing the long-term growth on 12-well MEAs, we obtained a complex network activity from Dopa.4U neurons which were similar to primary neurons. Dopa.4U neurons developed coordinated population bursts between 14 and 21 days in vitro (Fig. 1), activity was comparable to mouse cortical cultures in absolute values (data not shown) and also shown by the reduction of coefficient of variation over time and in the network for spike and bursts rate indicating a higher regularity and synchronicity, respectively (Fig. 2).
Both, primary mouse and human neuron cultures were treated acutely with 0.1-10 µM of the known pro-convulsive compounds Picrotoxin (PTX, GABA-A receptor blocker), N-Methyl-D-aspartate (NMDA, NMDA receptor agonist) and 4-aminopyridone (4-AP, Kv1 channel blocker). We have observed concentration-dependent increase of activity (spike rate and burst rate) for PTX, NMDA and 4-AP in both culture types (Fig. 3, 4). Some differences are observed between the two culture types: Interestingly, PTX increased spike and burst rate at 1 µM whereas cortical cells were excited at 10 µM. NDMA-mediated excitotoxicity (loss of activity, increase of interburst interval) was observed at 10 µM in DOPA.4U which was not the case yet in cortical neurons. NMDA induced a loss of spike in burst in both culture types. Here, cortical cultures showed a stronger increase of spiking variability over the network (CVnet) and in time (SD) compared to human DOPA.4U neurons. DMSO did increase activity, synchronicity or regularity significantly (data not shown).
Complexity of activity patterns and network communication are required to investigate compound effects on human neuronal networks using MEAs. Here, we present a human iPSC-derived neuron culture with strong activity development into a relatively mature functional status which is comparable with primary mouse neurons. Although not tested at the peak activity which could be obtained, known pro-convulsive compounds PTX, NMDA and 4-AP affect DOPA.4U activity significantly. Noteworthy, in a variety of parameters PTX, NMDA and 4-AP were partly more potent in terms of increasing activity and the putative excitotoxicity-mediated loss of activity effects than those recorded on primary mouse cortex cultures.
The comparison of these effects in a time-dependent (maturity-dependent) manner needs further investigation. Also, comparison with different hiPSC-neuronal cultures is required to improve interpretation and reliability for using human iPSC neurons for drug screening and safety assessment of compounds.
In conclusion, a systematic comparison of seizure liability in different hiPSC-derived neurons with primary rodent cortex cultures is a promising approach to identify the most relevant differentiation protocol for hiPS cells needed to improve predictability, sensitivity and specificity of future safety in vitro tests as well as to reduce the number of test animals for safety assessment (3Rs).
This study was partly supported by NC3R/UK CrackIT NeuraTect Program.
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
MEA Meeting 2016
(2016). Comparing drug-induced seizure-liability in human iPSC-derived and primary mouse neurons grown on micro-electrode arrays.
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
22 Jun 2016;
24 Jun 2016.
Dr. Benjamin Bader, NeuroProof GmbH, Rostock, Germany, email@example.com