ELRIG Drug Discovery 2017 Oct 3-4 Liverpool UK Pharmacological Responses in Cultured Human ipsc Pharmacological evaluation derived Cortical Neurons using Multi-electrode Array in human ipsc-derived cortical and sensory neurons using high-throughput MEA system Department of electronics, Tohoku institute of Technology Ikuro Suzuki
Background Many neuronal phenotypes have been derived from human ipscs Human ips cell Neuron Normal neuron Glutamate GABA Dopamine Acetylcholine Alzheimer s Parkinson s Huntington s Rett Syndrome Epilepsy Disease model Application of these cells to toxicological assay and drug discovery
Background Cook et al., Nat. Rev. Drug Descov., 13, 419 431, 2014 Improvement of risk accuracy in preclinical studies is important issue. Human ipsc-derived neurons are expected as a new toxicological evaluation assay to replace animal experiments in preclinical studies.
Purpose Toxicological evaluation assay in human ipsc-derived neurons Today s topics MEA assay in CNS to predict the seizure risks MEA assay in PNS to predict the pain risks
0.1 mv Methods Features for MEA Electrophysiological methods High-throuput multi-electrode array system (Presto) Non invasive Real time Multi-point measurement of activity of cultured neurons Extracellular signals of action potentials 24 wells (384 electrodes) 16 electrodes/well Presto System High-sensitivity 384 electrodes 5 sec Low impedance electrode
Methods human ipsc-derived cortical neurons Human ipsc-derived Neural Stem Cells Human ipsc-derived Mature Astrocyte + Co-culture neurons with astrocyte
Colocalization of pre- and postsynaptic components Merge Hoechst 33258 β-tubulin Ⅲ Synaptophysin PSD95 PSD95/Synaptophysi n 50mm Colocalization was confirmed, and synapses had mainly formed around thick dendrites and the soma Odawara A, et.al, Sci Rep 6, 26181 (2016).
Morphology (Pyramidal-like morphology) Pyramidal-like morphology with thick apical and basal dendrites β-tubulin Ⅲ Synaptophysin 50mm β-tubulin Ⅲ Synaptophysin Hoechst 33258 50mm These neurons were similar in appearance to cerebral cortical neurons in vivo Odawara A, et.al, Sci Rep 6, 26181 (2016).
0.1 mv 0.1 mv Pharmacological responses hipsc-derived neurons on the MEA β-tubulin Ⅲ Synaptophysin PSD95 GABA-A receptor Kainate Before Bicuculline Bicuculline Kainic acid Kainic acid Pharmacological responses Befo Bicuculli Kainic Kainic ac NMDA AP-5 AP5 AP AMPA CNQX CNQ 55 sec Before Before Bicuculline Bicuculline Bicuculline Bicuculline Kainic acid Kainic acid Kainic Kainic acid AP-5 AP5 AP-5 AP5 CNQX CNQX 55 sec We confirmed electrophysiological responses of typical GABA and glutamate receptors 10 msec Odawara A, et.al, Sci Rep 6, 26181 (2016).
Counts No. of spikes (%) Spikes/bin No. of spikes( %) Plasticity in hipsc-derived neurons (a) 34ch Before (b) 250 200 HFS (c) LTP LTP 54ch 34ch After 150 100 50 before after LTD 54ch (d) (a) 6 5 4 3 2 1 0 45ch 57ch 45ch 10 LTP 50 100 150 200 250 300 57ch Before After Change rate (%) (b) (e) (e) 0 0 15 5 30 10 45 15 60 20 75 25 90 30 105 35 120 40 150 125 100 75 50 25 0 Time (min) HFS Time (ms) 0 15 5 30 10 45 15 60 20 75 25 90 30 105 35 120 40 Time (min) before after (c) LTD Before After (d) 12 We reported that human ipsc-derived cortical neurons also have the plasticity. 1600 LTD 10 10 8 8 6 6 4 4 2 2 0 0-2 -2-4 -4-6 -6-8 -10-8 Before Odawara AfterA, et.al, BBRC 496, 856 (2016). -10
Induction of epileptiform activity Major toxicity of new drugs in CNS is seizure-like firings Convulsant drugs 4-Aminopyridine (4-AP): K + channel antagonist Pilocarpine : Muscarine receptor agonist Chlorpromazine : D 2 receptor antagonist Pentylenetetrazole (PTZ): GABA-A antagonist Anti-convulsant drug Phenytoin: Na + channel antagonist Negative control Acetaminophen: analgesic effect
Results:Raster plots in 4-AP administration 0 µm 0.3 µm 1 µm 3 µm 10 µm 30 µm 4-Aminopryridine 1 min The number of synchronized bursts were increased in a concentration-dependent manner. 1s
Movie at before and 4-AP 10 μm administration
Results:Raster plots in pilocarpine administration 0 µm 0.3 µm 1 µm 3 µm 10 µm 30 µm Pilocarpine The number of synchronized bursts were also increased in a concentration-dependent manner at pilocarpine administration 1 min
Results:Raster plots in Chlorpromazine administration 0 µm 0.1 µm 0.3 µm 1 µm 3 µm 10 µm Chlorpromazine 1 min The number of synchronized bursts were also increased up to 3 μm and disappeared at 10 μm administration.
Results:Raster plots in PTZ administration 0 µm Pentylenetetrazole 1 µm 10 µm 100 µm 1000 µm 1 min The number of synchronized bursts were not changed. The duration of syncoronized burst were increased in a concentration-dependent manner.
No. of burst v.s. 0µM (%) No. of burst v.s. 0µM (%) No. of total spikes v.s. 0µM (%) No. of total spikes v.s. 0µM (%) Results:Firing analysis in 4-AP, pilocarpine, chlorpromazine, and PTZ administration Number of synchronized burst Total spikes 700 250 120 400 200 100 150 80 60 100 40 50 20 0 0 4-AP Pilocarpine Chlorpromazine PTZ 4-AP Pilocarpine Chlorpromazine PTZ 4-AP Pilocarpine Chlorpromazine PTZ PTZ 4-AP Pilocarpine Chlorpromazine PTZ Number of synchronized bursts were increased at 4-AP, pilocarpine and chlorpromazine administration. On the other hand, number of synchronized bursts were decreased at PTZ administration.
Duration v.s. v.s. 0µM 0µM (%) (%) No. No. of of spikes in a burst v.s. v.s. 0µM 0µM (%) (%) Results:Firing analysis in 4-AP, pilocarpine, chlorpromazine, and PTZ administration Duration of synchronized burst Spikes in a synchronized burst 160 140 140 120 120 100 80 60 100 80 60 40 40 20 20 0 0 4-AP 4-AP Pilocarpine 4-AP Pilocarpine Chlorpromazine PTZ 4-AP Pilocarpine Chlorpromazine PTZ We detected the effects of four convulsant durgs in human ipsc-derived neurons and found that the responses of PTZ is different from 4-AP, pilocarpine and chlorpromazine.
Total spikes v.s. before (%) Results:Raster plots in phenytoin administration Before 0μM Phenytoin 120 Total spikes 1μM 100 80 ** 3μM 60 10μM 30μM 100μM 40 20 0 ** before 0uM 1uM 3uM 10uM 30uM 100uM The number of synchronized bursts disappeared at 100 μm phenytoin administration. We confirmed the responses of Na + channel blocker.
Total spikes v.s. before (%) Results:Raster plots in acetaminophen administration Acetaminophen Total spikes 0 µm 180 160 1 µm 140 120 3 µm 100 10 µm 80 60 30 µm 40 20 100 µm 1 min 0 0uM 1uM 3uM 10uM 30uM 100uM The number of synchronized bursts and total spikes were not changed in a concentration-dependent manner at acetaminophen administration
Summary 1 The induction of epileptiform activity by 4-AP, pilocarpine, chlorpromazine and PTZ and the suppressive effects by phenytoin were detected in hipscderived cortical neurons made by Axol bioscience. High-throughput MEA system in cultured hipsc-derived neurons and our analysis tools are useful for toxicological evaluation in human CNS.
Peripheral nervous system Sensory neuron Waxman and Zamponi, 2014, Nat Neurosci, modyfied Human ipsc-derived sensory neurons are suitable to toxicological assay for pain
Methods human ipsc-derived sensory neurons Human ipsc-derived sensory neuron progenitors Dr Edward Emery (University College London).
We confirmed the typical human sensory marker. Results:Sensory marker expression 8 weeks Nav 1.7 β-tubulin Ⅲ Hoechst 33258 Merge TRPV1 TRPA1
Electrode number Results:Capsaicin Capsaicin DMSO 0.001%, Tween80 0.001% Capsaicin 100 nm hipsc-derived Sensory neurons on the MEA 2 3 4 5 6 7 Time (min) 6 5 4 3 2 1 0-5 -4-3 -2-1 0 1 2 3 4 5 time (s) We confirmed that TRPV1 channels was working electrophysiologically.
Results:Temperature change The responses against temperature change From 37 to 46 37 43 37 38 40 42 44 46 It was consistent with the activation temperature of TRPV1 channels
Results:Menthol and AITC(wasabi) Electrode number Electrode number Menthol AITC(wasabi) TRPM8 Before Menthol 100μM TRPA1 Before 6 200ms 40mV 6 5 5 4 4 3 3 2 2 1 1 0-5 -4-3 -2-1 0 1 2 3 4 5 Time (s) 0-5 -4-3 -2-1 0 1 2 3 4 5 Time (s) We also confirmed TRPM8 and TRPA1 channel was working electrophysiologically.
Percentage of neurons (%) Results:Classification by electrophysiological responses Human ipsc-derived sensory neurons were classified into 27 types depending on physiological responses against 3 compounds (Capsaicin, Menthol, and AITC). 25 20 Human ipsc-derived sensory neurons (n=790) Rat sensory neurons (n=345) 15 10 5 0 Cap Men AIT + + + + + + + + + - - - - - - - - - ± ± ± ± ± ± ± ± ± + + + - - - ± ± ± + + + - - - ± ± ± + + + - - - ± ± ± + - ± + - ± + - ± + - ± + - ± + - ± + - ± + - ± + - ± Percentage of positive responses against capsaicin were high both hipsc-derived neurons and rat DRG neurons. hipsc-derived sensory neurons have a variety of physiological properties and resemble to rat DRG neurons.
Electrodes AWSDR Total spikes v.s. Before (%) Results:Anti-cancer drug(oxaliplatin and Vincristine) AWSDR Electrodes 16 8 Oxaliplatin 0 nm 1 nm 10 nm 100 nm 1000 nm 4500 4000 3500 3000 2500 2000 1500 1000 500 n=4 120 100 80 60 40 20 Normalized firing rate(%) 1 0 0 Vincristine 1 min 0 nm 10 nm 100 nm 1000 nm 16 8 1 MEA measurement in hipsc-derived sensory neurons is useful to pain evaluation of anti-cancer drugs 1 min 1 min
Electrodes AWSDR Results:Pain relief drug Acetylsalicyclic Acid (Aspirin) 0 µm 1 μm 30 μm 100 μm 300 μm 1000 μm 16 8 1 1 min We also confirmed no responses to pain relief asprin in cultured hipsc-derived sensory neurons.
Summary 2 Humen ipsc-derived sensory neurons (Axol Bioscience) show the expression of typical sensory neural marker Nav1.7, TRPV1, and TRPA1. We detected the physiological responses to temperature change, capsaicin, menthol, and AITC. Human ipsc-derived sensory neurons were classified into 27 types depending on physiological responses against 3 compounds. We detected the responses to anti-cancer drugs oxaliplatin and vincristine in cultured hipsc-derived sensory neurons. We will investigate the mechanism of action in the responses at anti-cancer drug administration. MEA measurement in cultured hipsc derived sensory neurons are suitable to toxicological assay for pain in peripheral nervous system.
Acknowledgement Tohoku Institute of technology Suzuki lab A. Odawara, N. Matsuda, Y. Ishibashi, R. Yokoi, C. Iemura Alpha Med Scientific (Japan). R. Yamazaki, H. Jiko, M.Fukuda M. Trujillo, R.Arant, G. Cheng Axol Bioscience (UK). Zoe Nilsson, Priyanka Dutta, Nick Clare, Sanj Kumar