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Neurotoxicity assessment: Comparison between SH-SY5Y and iPSC-derived cells

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As shown by AstraZeneca in Nature reviews*, one third of the safety failures is linked to CNS toxicity during the clinical trials of drugs. Therefore, relevant in vitro human model is needed to detect early neurotoxicity of drug candidates. In this study, sensitivity of iPSC-derived neuronal cells to 32 compounds is compared to the sensitivity of SH-SY5Y cells. Two types of iPS-derived cells are tested: central nervous system cells (CNS.4U™ cells) and peripheral nervous system cells (PERI.4U™ cells) from Ncardia. Toxic effects are then measured by HCS cell imaging.

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Neurotoxicity assessment: Comparison between SH-SY5Y and iPSC-derived cells

  1. 1. Neurotoxicity assessment: Comparison between SH-SY5Y and iPSC-derived cells De Conto V. 1, Vandenhaute E.1*, Fouassier A.2, Hess D.2, Palm T.2, MAUBON N.1 1HCS Pharma, 250 rue Salvador Allende, 59120 Loos, France 1 2Ncardia, Nattermannallee 1, Bldg S20, 50829 Cologne, Germany *elodie.vandenhaute@hcs-pharma.com Abstract As shown by AstraZeneca in Nature reviews (Cook et al., 2014), one third of the safety failures is linked to CNS toxicity during the clinical trials of drugs . Therefore, relevant in vitro human model is needed to detect early neurotoxicity of drug candidates. In this study, sensitivity of iPSC-derived neuronal cells to 32 compounds is compared to the sensitivity of SH-SY5Y cells. Two types of iPS-derived cells are tested: central nervous system cells (CNS.4U™ cells) and peripheral nervous system cells (PERI.4U™ cells) from Ncardia. Toxic effects are then measured by HCS cell imaging. Methods Conclusions & Perspectives - CNS4U™ and PERI4U™ seem to be interesting and valuable alternatives to neuronal cell lines like SH-SY5Y since they seem to be more sensitive to neurotoxicity compared to SH-SY5Y - Presence of astrocytes in the cellular mixture CNS.4U™can impact on neuronal responses and survival of cells - Apart from toxicity studies, iPSC-derived neuronal cells are of great interest in pathophysiological studies (e.g. neurodegenerative diseases) - The next step will be to perform these assays in 3D culture. Cell culture: SH-SY5Y were grown at 37°C with 5% CO2 in MEM/F12 supplemented with 10% FBS, 1% L-glutamine, 1% non essential amino-acids and 1% penicillin/streptomycin. For all assays, cells were plated in 384-well plates (2,500 cells per well) in differentiation media (containing 25nM staurosporine). iPSC-derived cells were seeded in 384-well plate immediately after thawing (10,000 cells per well) in complete Neuro.4U™ media (Ncardia). Cell treatment: Cells were treated three days after seeding with a library of 32 molecules (0.03-30µM), considered as non- toxic compounds, generally toxic but non-neurotoxic compounds and neurotoxic compounds, during 48 hours (Tong et al., 2017). Microscopy: Cells were stained with anti-β3-tubulin antibodies and Hoechst (DNA staining). Imaging was done with ImageXpress Micro Confocal Imaging System, and image analysis was performed with MetaXpress software (Molecular Devices) to quantify cell viability and neurite outgrowth. Number of cells and neurite outgrowth were normalized to the values for control cells treated with 0.5% DMSO (vehicle). Neurite outgrowth Results To get this poster, please flash the QR- code. You can use the I- NIGMA application from your store SH-SY5Y, CNS.4U and PERI.4U cells , stained with Hoechst (blue) for nuclei and for β3-tubulin (green).Cell viability and neurite outgrowth were measured after 48h of incubation with the different compounds or with the vehicule alone for control cells (DMSO 0.5%). Scale bar =200µm. Control CNS.4U™Control SH-SY5Y Control PERI.4U™ SH-SY5Y – Colchicine30 µM CNS.4U™ – Colchicine30 µM PERI.4U™ – Colchicine 30 µM Viability SH-SY5Y CNS.4U PERI.4U SH-SY5Y CNS.4U PERI.4U Amoxicillin = = = Colchicine ++ ++ ++ Celecoxib +  + Dexamethasone = = + Diphenhydramine = = = Dipyridamole +  = D-Sorbitol = = = Imipramine hydrochloride + + + L-Ascorbic acid =  = Lead chloride + ++ + Metformin hydrochloride =  = Lidocaine ++ ++ + Nadolol = = = t-Retinoic acid + = + Saccharin sodium salt  = = Nocozadole ++ ++ ++ Acetaminophen +  = Paclitaxel + + + Indomethacin +  = Suramin = + + Quinidine + + + Tamoxifen + ++ ++ Troglitazone + + + Methylmercury chloride ++ ++ ++ Terodiline + + + Vinblastine sulfate ++ ++ ++ 6-Hydroxydopamine + + ++ Vincristine sulfate ++ ++ ++ Acrylamide + ++ ++ Chloroquine + + + Cisplatin + + + Sodium orthovanadate + + + Legend: = No effect ; + Neurotoxic effect ; ++ High neurotoxic effect ;  Increased number of cells PERI.4U™ – Nadolol 30 µMCNS.4U™ – Nadolol 30 µMSH-SY5Y – Nadolol 30 µM SH-SY5Y – Suramin 30 µM CNS.4U™ – Suramin 30 µM PERI.4U™ – Suramin 30 µM NadololSuraminColchicine SUMMARY OF THE RESULTS: NEUROTOXIC EFFECT OF 32 COMPOUNDS References: - Cook D, Brown D, Alexander R, March R, Morgan P, Satterthwaite G, Pangalos MN (2014) Lessons learned from the fate of AstraZeneca’s drug pipeline: a five-imensional framework. Nat Rev Drug Discov. 13(6):419-31. - Tong Z.-B., Hogberg H., Kuo D., Sakamuru S., Xia M., Smirnova L., Hartung T., Gerhold D. (2017) Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening. Journal of Applied Toxicology. 37(2):167-80. Non-toxicNon-neurotoxicNeurotoxic Neurotoxic

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