title: First steps in establishing a developmental toxicity test method based on human embryonic stem cells.
abstract: The use of embryonic stem cells is currently the most promising approach to assess developmental toxicity in vitro. In addition, the possibility of using human embryonic stem (hES) cells will increase safety of consumers and patients as false classification of substances due to inter-species variations can be avoided. One validated test based on murine embryonic stem cells, the embryonic stem cell test (EST), consists of following endpoints: IC(50) values of fibroblasts and embryonic stem cells as well as the inhibition of differentiation of mES cells into cardiomyocytes. As a follow up of its successful validation study we established a cytotoxicity assay based on hES cells and human fibroblasts employing two developmental toxicants: 5-fluorouracil (5-FU) and all-trans retinoic acid (RA). The results were compared to historical data from the EST. For 5-FU, no significant differences were obtained between the different cell lines. However, for RA, both test systems produced higher IC(50) values for the fibroblasts than for the stem cells, which is a well-known effect of developmental toxicants. Moreover, the reliability and relevance of several marker genes as possible toxicological endpoints were tested. During early differentiation Oct-4, hTert and Dusp6 showed the most reliable results. Brachyury and GATA-4 were found to be best suited to monitor cardiac differentiation. The late cardiac marker gene TNNT2 demonstrated significant results until day 18. Therefore, these marker genes have the highest potential to serve as endpoints for a developmental toxicity test.
authors.affiliation: ECVAM, Joint Research Centre, Institute for Health and Consumer Protection, Via E. Fermi 1, 21020 Ispra (VA), Italy. sarah.adler@cellartis.com
authors.forename: Sarah
authors.initials: S
authors.lastname: Adler
authors.forename: Cristian
authors.initials: C
authors.lastname: Pellizzer
authors.forename: Lars
authors.initials: L
authors.lastname: Hareng
authors.forename: Thomas
authors.initials: T
authors.lastname: Hartung
authors.forename: Susanne
authors.initials: S
authors.lastname: Bremer
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000633:toxicity; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D023381:Endpoint Determination; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005472:Fluorouracil / Q000008:administration & dosage / Q000633:toxicity*; D006801:Humans; D066298:In Vitro Techniques; D020128:Inhibitory Concentration 50; D051379:Mice; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*; D014212:Tretinoin / Q000008:administration & dosage / Q000633:toxicity*
chemical_list: D000970:Antineoplastic Agents; D015415:Biomarkers; D014212:Tretinoin; D005472:Fluorouracil