Abstract
Toxicity is generally referred to as the degree to which a substance can damage an organism. According to numerous reports, determination of toxicity through an in vitro system is an economical and ethical method, compared with an in vivo system using animals. The hES cells can differentiate into three germ layers and the unique feature of hES cells from the three germ layers via the EB formation adds good insight in understanding human developmental biology in vitro. Evaluation of developmental toxicity of a compound on early embryo states can therefore be based on its effect on EB formation. In this study, we used mouse embryonic stem cells (mESC) to investigate the effects of several developmental toxic chemicals on the formation of EBs. We used the EB hanging drop method and tested five toxic chemicals; cytosine arabinoside, dexamethasone, hydroxyurea, indomethacin, 5-fluorouracil, and two negative controls; ascorbic acid and penicillin G. We demonstrated a significant reduction of EB size after treatment with a high dose of each chemical. We evaluated cell toxicity by performing measurements of cell viability after treatment with each chemical. Expression of apoptosis-related genes (p53, Sirt1, p21, Puma, Noxa, Mdm2) and pluripotency marker genes (Oct4, Nanog, Sox2, ZFP206) and differentiation marker genes, such as endoderm (HNF4, AFP), mesoderm (T-brachy), and ectoderm (Pax6) were determined by quantitative real-time PCR. The chemicals induced abnormal differentiation and apoptosis. We confirmed apoptotic positive cells by TUNEL assay. In addition, we also demonstrate the reduction of the size of EBs through expression of apoptosis-related marker genes (p53, Caspase-3, PARP) and necrosis-related marker gene (HMGB1). The results obtained demonstrate that EBs can be used as an in vitro model for testing developmental toxicity.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.