206 ESTIMATION OF EMBRYO DEVELOPMENTAL TOXICANTS IN HUMAN EMBRYONIC STEM CELL

An embryonic stem cell test (EST) has been developed to evaluate the embryotoxic potential of chemicals with an in vitro system. In the present study, novel methods to screen toxic chemicals during the developmental process were evaluated using undifferentiated human embryonic stem (hES: H9) cells. hES H9 cells were obtained from WiCell Research Institute, Inc., (Madison, WI, USA) and cultured according to the manufacturer's protocol. The cells were cultured on mouse embryonic fibroblast (mEF) cells in hES cell medium composed of DMEM/F12 supplemented with 20% defined fetal bovine serum (FBS), 1% minimal essential medium (MEM) nonessential amino acids, 0.1 mM b-mercaptoethanol, 2 mM L-glutamine, 50 U mL–1 penicillin/streptomycin, and 4 ng mL–1 human recombinant basic fibroblast growth factor. By using surface marker antigens (SSEA-4, TRA-1-60, and TRA-1-81), we confirmed undifferentiated conditions of the used hES cells by immunocytochemistry. We assessed the developmental toxicity of well-known embryotoxic chemicals (cytosine arabinoside, 5-fluorouracil, hydroxyurea, and indomethacin) by expression of pluripotent ES cell markers (OCT-4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2(SFRP2), teratocarcinoma-derived growth factor 1 (TDGF1), and phosphatase, and tensin homologue (PTEN)) in different concentrations for up to 7 days. Expression of pluripotent ES cell markers were determined by quantitative real-time PCR. Data are presented as the mean ± standard error of the mean (s.e.m.), and were analysed with a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. P-values <0.05 were considered to be statistically significant. Although expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers. Most of the pluripotent ES cell markers were down-regulated in dose-dependent manner following treatment with embryotoxic chemicals. After treatment with 5-fluorouracil, indomethacin, and penicillin G, we observed a remarkable convergence in the degree of up-regulation of development, cell cycle, and apoptosis-related genes by gene expression profiles using Affymetrix GeneChips (Santa Clara, CA, USA). Taken together, these results suggest that embryotoxic chemicals have cytotoxic effects, and modulate the expression of ES cell markers as well as development-, cell cycle-, and apoptosis-related genes that have pivotal roles in undifferentiated hES cells. Therefore, we suggest that hES cells may be useful for testing the toxic effects of chemicals that could affect the embryonic developmental stage.