Jung Eui-Man, Choi Yeo-ul, Kang Hong-Seok, Yang Hyun, Hong Eui-Ju, An Beum-Soo, Yang Jun-young, Choi Ki Hwan, Jeung Eui-Bae
Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea.
J Appl Toxicol. 2015 Feb;35(2):205-18. doi: 10.1002/jat.3010. Epub 2014 Apr 16.
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) cells. 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 embryotoxic chemicals, 5-fluorouracil, indomethacin and non-embryotoxic penicillin G in different concentrations for up to 7 days. While expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers, such as OCT-4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2 (SFRP2), teratocarcinoma-derived growth factor 1 (TDGF1), and phosphatase and tensin homolog (PTEN). Most of the pluripotent ES cell markers were down-regulated in a dose-dependent manner after 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 an Affymetrix GeneChips. 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 impact the embryonic developmental stage.
已开发出一种胚胎干细胞试验（EST），用于通过体外系统评估化学物质的胚胎毒性潜力。在本研究中，使用未分化的人类胚胎干细胞（hES）评估了在发育过程中筛选有毒化学物质的新方法。通过使用表面标志物抗原（SSEA-4、TRA-1-60和TRA-1-81），我们通过免疫细胞化学证实了所用hES细胞的未分化状态。我们评估了胚胎毒性化学物质5-氟尿嘧啶、吲哚美辛和非胚胎毒性青霉素G在不同浓度下长达7天的发育毒性。虽然表面标志物的表达没有受到显著影响，但胚胎毒性化学物质影响了它们对多能性ES细胞标志物的反应，如OCT-4、NANOG、内皮素B型受体（EDNRB）、分泌型卷曲相关蛋白2（SFRP2）、畸胎瘤衍生生长因子1（TDGF1）和磷酸酶及张力蛋白同源物（PTEN）。在用胚胎毒性化学物质处理后，大多数多能性ES细胞标志物以剂量依赖的方式下调。在用5-氟尿嘧啶、吲哚美辛和青霉素G处理后，我们使用Affymetrix基因芯片通过基因表达谱观察到发育、细胞周期和凋亡相关基因上调程度的显著趋同。综上所述，这些结果表明胚胎毒性化学物质具有细胞毒性作用，并调节ES细胞标志物以及在未分化hES细胞中起关键作用的发育、细胞周期和凋亡相关基因的表达。因此，我们认为hES细胞可能有助于测试可能影响胚胎发育阶段的化学物质的毒性作用。
