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Home , Histamine agonist, Muscarine

Application of Rapid iPSC Differentiation 0015 to Assays Rie Yamoto1*, Hikaru Watanabe2, Manabu Seo3, Toshihiko Hosoya1 1Drug Discovery Business Department, Healthcare Business Group, RICOH COMPANY, Ltd. 2Biomedical Research Department, Healthcare Business Group, RICOH COMPANY, Ltd. 3Elixirgen Scientific, Inc. *e-mail; [email protected] Introduction Materials and Methods Summary of compounds TM TM The Quick-Tissue technology is a Cells: Excitatory neurons were generated using the Quick-Tissue Compound Function Conc. (μM) transcription factor-based method for technology from iPSCs originating from a healthy donor (Elixirgen Scientific, GABA GABA 0.1, 1 rapid differentiation of stem cells Inc. #EX-SeV-CW50065). Primary human astrocytes were obtained from Bicuculline1 3, 30 (iPSCs or ESCs) into desired cell types. Thermo Fisher Scientific Inc. Gabazine1 10, 100 2 GABA(A) antagonist The method generates pure Pentylentetrazole 100, 1000 Spheroid culture: 96-well plates were seeded with the neurons and pre- 1 populations of differentiated cells 1, 10 cultured astrocytes at the density of 8,000 cells/well. Plates were cultured Glutamate AMPA/NMDA agonist 10, 100 within 1-2 weeks. in the BrainPhys medium (STEMCELL technologies Inc.) for 6 weeks. Kainic Acid Kainate agonist 1, 10 NMDA NMDA agonist 10, 100 To investigate the suitability of Quick- Ca2+ imaging: Spheroids were loaded with a fluorescent Ca2+ reporter dye Tissue™ neurons to toxicological CNQX AMPA/Kainate antagonist 10, 100 (MOLECULAR DEVICES, LLC.). Fluorescence intensity (FI) was measured D-AP5 NMDA antagonist 3, 30 assays, we analyzed their with the FDSS6000 system (Hamamatsu Photonics K.K.). responses Dopamine 10, 100 pharmacological characteristics using were analyzed by determining the change in the number of peaks of Ca2+ 10, 100 2+ Dopamine D2 antagonist Ca imaging. oscillation and the ratio of FI before and after the drug application. Haloperidol 1, 10 5-HT Serotonin agonist 1, 10 Statistical significance of the comparison to 0.1 % DMSO was determined Serotonin H1/2 agonist 3, 30 1-2 weeks by the Wilcoxon rank sum test. Buspirone Mono amine blocker 3, 30 1, 10 Muscarine agonist Drug application Drug application Pilocarpine2 1, 10 Muscarine antagonist 1, 10 Strychnine1 Nicotine/Gly antagonist 3, 30 Histamine agonist 3, 30 Ketotifen1 1, 10 Histamine Diphenhydramine1 1, 10 4-Aminopiridine 3, 30 Potassium Terminally the number of peaks the number of peaks FI averaged FI averaged Linopirdine 10, 100 differentiated for 10 min for 10 min for the last 1min for the first 1min Carbamazepine3 10, 100 neuron turnaround Phenytoin3 Sodium channel blocker 10, 100 (weeks) Valproic acid3 100, 1000 change in the number of peaks 2 Source; FI Ratio (after/before) Enoxacin Antibacterial agent 1, 10 Company websites (after-before) Analysis method 1; Convulsant, 2; Epileptogenic, 3; Antiepileptic Results Ca2+ response to major pharmacological compounds (a) (b) GABA Kainic acid D-AP5 * *: p<0.05 1 10 30 * μM μM μM

0.1 1 3 μM μM μM DMSO Dopamine 5-HT Histamine

100 10 30 μM μM μM * * * * *

10 1 3 μM μM μM

Diphenhydramine Linpirdine Carbamazepine

10 100 100 (c) μM μM μM * * 1 10 10 * *: p<0.05 μM μM μM * * * DMSO

0.1 DMSO %

5 min (a) Waveforms of Ca2+ oscillation before and after the ** * * application of that affect the 6 major types of receptors. * (b) Change in the number Ca2+ oscillation peaks. The peaks were counted during 10 min before and after the drug application (n=6). (c) Ratio of the fluorescence intensity (FI) measured during 1 min before and after the drug application (n=6).

Summary of drug response ReceptorsDrugs Response GABA Agonist all not determined Summary and Conclusions Functional receptors Antagonist GABAA ×  Pharmacological analyses of Quick-Tissue™ neurons Glutamate Agonist AMPA/NMDA 〇 Excitatory Confirmed by the responses to the suggest the presence of functional receptors for all the 6 Kainate 〇* Excitatory ; Glutamate, Dopamine, NMDA 〇 Excitatory major and functional channels for two Serotonin, Cholin, and Histamine Antagonist AMPA/Kainate 〇* cations. The results also suggest synaptic connections for NMDA 〇 Functional ion channels Dopamine Agonist all 〇* Inhibitory Glutamate, Serotonin, Cholin, and Histamine. The Antagonist D2 × Confirmed by the responses to the neurons also responded to convulsant, epileptogenic, and + + Serotonin Agonist all 〇* Inhibitory blockers; K and Na antiepileptic drugs. Reuptake blocker 〇 Excitatory 2+ Cholin Agonist Muscarine 〇 Inhibitory Functional Synapses  The fluorescent intensity (FI) and the number for Ca Antagonist Muscarine 〇 Confirmed by the responses to the oscillation peaks responded to drugs and thus likely Nicotine/Gly 〇 antagonists; Glutamate, Serotonin, constitute reliable drug response markers. Histamine Agonist all 〇* Excitatory Cholin, and Histamine Antagonist H1 〇*  These results suggest Quick-TissueTM neurons are suited Ion channel blocker Potassium 〇* to neurotoxicity assays and drug screenings. Sodium 〇* *: p<0.05 The concentration of GABA in this test was lower than IC50 COI Disclosure Information We have the following financial relationships to disclose. value (2.8 μM) and the data was removed from the analysis. Research funds from: Elixirgen Scientific, Inc. copyright © 2020 RICOH COMPANY, Ltd. All rights reserved.