Safety Evaluation Study Using Embryonic Stem (ES) Cells

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1 Safety Evaluation Study Using Embryonic Stem (ES) Cells Sumitomo Chemical Co., Ltd. Environmental Health Science Laboratory Nobuyuki HORIE Hashihiro HIGUCHI Satoshi KAWAMURA Koichi SAITO Noriyuki SUZUKI Embryonic stem (ES) cells are pluripotent stem cells that have the capacity for self-renewal and multilineage differentiation, and they have recently started to be used in the safety evaluation of chemicals. The novel in vitro embryotoxicity test (EST) that utilized the differentiation ability of mouse ES cells into cardiomyocytes was established in Germany. We could obtain results which were equal to the validation study performed in Europe and will apply the test system to a preliminary assessment of new chemicals. In addition, we have participated in one of the national projects to improve this test system to make it much simpler and more precise. Embryonic stem cell ; ES Blastocyst ; Inner cell mass Fig ) ) ES 2 Fig. 2 Zygote 2-cell 4-cell 8-cell Inner cell mass ES Cells Blastocyst Morula Fig. 1 Generation of the ES cell line 2007-

2 Self-renewal ES cell Differentiation cardiomyocyte neuron skeleton Fig. 2 Pluripotency of ES cells ES ES ES ES LIF leukemia inhibitory factor ; ES ES Fig. 3 Undifferentiated mouse ES cells ES ES ES Fig. 3 : ES in vitro in vitro in vitro 1991ECVAM European Centre for the Validation of Alternative Methods 1993ICC- VAM Interagency Coordinating Committee on the Validation of Alternative Methods 3 OECD Organization for Economic Cooperation and Development Table JaCVAM Japanese Center for the Validation of Alternative Methods 2007-

3 Table 1 Toxicity studies Skin Corrosion Phototoxicity Skin Absorption Eye Irritation/Corrosion Acute Toxicity Repeated Dose Toxicity Carcinogenicity Teratology Reproductive Toxicity Evaluations of alternative (non-animal) test by OECD OECD Test Guidelines (In vitro) No.430 In vitro Transcutaneous Electrical Resistance No.431 Human Skin Model Test No.435 In vitro Membrane Barrier Method for Skin Corrosion No.432 In vitro 3T3 NRU Test No.428 In vitro Method 1961 in vitro in vitro Table 2 ES EST : Embryonic Stem cell Test Micromass culture Whole embryo culture 3) ES in vitro Table 2 In vitro embryonic toxicity studies 1. Mammals Study Material Index Reference Cell Lines Cells Primary Cultures Organs Whole embryo culture MOT Assay HEPM Assay Gap junction Embryonic Stem cell test Micromass culture Palate culture Limb bud culture Whole embryo culture Ascitic mouse ovarian tumour cells Human embryonic palatal mesenchyme cells Chinese hamster ovary cells Mouse enbryonic stem cells Mouse fibroblast cells Rat embryo (limb bud) Mouse embryo (palate) Mouse embryo (limb bud) Rat embryo Inhibition of cell attachment to lectin-coated sufases Cell proliferation Inhibition of gap junction Cardiomyocyte differentiation Cell proliferation Cartilage differentiation Cell proliferation Fusion of secondary palate Cartilage differentiation Morphology Morpholgenetic differentiation Braun et al., 1982 Pratt et al., 1985 Trosko et al., 1982 Spielmann et al., 1997 Flint et al., 1984 Shiota et al., 1990 Freedman et al., 1982 Schmid et al., Non-mammals (1970s 1980s) (1) Vertebrates Fish, Avian or Frog embryo (2) Invertebrates Drosophila, Hydra, Sea urchin 2007-

4 ESTES BfRDr.H.Spielmann 4) EST 2 in vitro ESTES 2ES D3 Balb/c 3T3 1ES 2ES Fig. 5 ES ES cells 3T3 cells Fig. 5 (Trypsinize) 3days medium exchange 2days medium exchange 5days Cytotoxicity Assay (ES and 3T3 cells) (500cell/well) 10days Detection of viable cells using a Microtiter plate reader 1 Fig. 4 ES embryoid body ID50IC50 ES 50 (Trypsinize) Hanging drop (750cell/drop) Embryoid Body ES cells (3days) Suspension culture Adhesive culture Beating! (2days) (5days) Cardiomyocytes Fig. 4 Differentiation into cardiomyocyte 2007-

5 Function I IC50 3T3 ID log(ic50 3T3) log(ic50 D3) IC50 3T3 ECVAM IC50 3T3 ID50 Function II 3.65 log(ic50 3T3) log(ic50 D3) ESAC ECVAM Scientific IC50 3T3 Advisory Commitee IC50 3T3 ID50 Function III log(ic50 3T3) 1.92 log(ic50 D3) EST IC50 3T3 IC50 : 5 cytotoxic concentration, ID50 : 5 differentiation concentration 3T3 : mouse fibroblast cell line, D3 : mouse embryonic stem cell line OECD 2003 ECVAM Non- embryotoxic Weakly embryotoxic Strong embryotoxic Function I > Function II, Function III Function II > Function I, Function III Function III > Function I, Function II 5) Fig. 6 Prediction model of the EST 3 Fig. 63 ES EST ECVAM EST in vivo Fig. 7 in vivo (animal study) ECVAM Sumitomo Non- embryotoxic Weakly embryotoxic Strong embryotoxic Non- embryotoxic Weakly embryotoxic Strong embryotoxic Fig. 7 Nonembryotoxic 68% 16% % in vitro (EST) Weakly embryotoxic 32% 84% 6% 5 83% Strong embryotoxic 83% 10 in vivo = in vitro The validation results in ECVAM & Sumitomo-chemical EST in vivo Fig. 7 EST in vitroest ES NEDO

6 ESTES EST ES DNA ES ES in vitro ESTEST in vivo 2004ReProTect 6) OECD in vitro EST ES in vivo ES ES high-throughput EST Embryonic stem cell test BfRDr. H. Spielmann ZEBETDr. A. Seiler Dr. K. Hayess 1) M. J. Evans and M. H. Kaufman, Nature, 292, 154 (1981). 2) J. A. Thomson, J. Itskovitz-Eldor, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V.S. Marshall, and J. M. Jones, Science, 282 (6), 1145 (1998). 3) E. Genschow, H. Spielmann, G. Scholz, A. Seiler, N. Brown, A. Piersma, M. Brady, N. Clemann, H. Huuskonen, F. Paillard, S. Bremer, and K. Becker, ATLA, 30, 151 (2002). 4) H. Spielmann, I. Pohl, B. Doring, M. Liebsch, and F. Moldenhauer, In vitro Toxicology, 10 (1), 119 (1997). 5) S. Bremer, and T. Hartung, Current Pharmaceutical Design, 10, 2733 (2004). 6) L. Hareng, C. Pellizzer, S. Bremer, M. Schwarz, and T. Hartung, Reproductive Toxicology, 20, 441 (2005)

7 PROFILE Nobuyuki HORIE Koichi SAITO Hashihiro HIGUCHI Noriyuki SUZUKI Satoshi KAWAMURA 2007-