Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test.
The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a reliable, scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts. In order to identify more objective endpoints of differentiation other than the microscopic evaluation of "beating areas" and to adapt the EST to applications in high-throughput screening systems we improved and expanded the EST protocol by establishing molecular endpoints of differentiation. The quantitative expression of sarcomeric myosin heavy chain (MHC) and alpha-actinin genes under the influence of test compounds was studied employing intracellular flow cytometry. Strong embryotoxicants exerted a dose-dependent effect on both the expression levels of MHC and alpha-actinin and the differentiation into beating cardiomyocytes. Furthermore, quantitative FACS (fluorescence-activating cell sorting) analysis showed the same sensitivity for the classification of substances as the conventional endpoint but allowed a significant reduction of the test period. Within 7 days, maximal expression of sarcomeric marker proteins was observed. Our findings indicate that structural proteins of the sarcomere apparatus, alpha-actinin and myosin heavy chain (MHC), seem to be promising candidates to predict developmental toxicity in vivo from in vitro data. Thus, the improved EST holds promise as a new predictive screen for risk assessment with respect to developmental toxicity using stem cell technology and technological advances in the field of gene expression analysis.
Seiler
Andrea
A
National Center for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET), Federal Institute for Risk Assessment (BfR), Berlin 12277, Germany. seiler.zebet@bfr.bund.de
Visan
Anke
A
Buesen
Roland
R
Genschow
Elke
E
Spielmann
Horst
H
D000185:Actinin / Q000032:analysis / Q000096:biosynthesis; D000818:Animals; D041702:BALB 3T3 Cells; D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002469:Cell Separation; D003114:Colony-Forming Units Assay / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D005472:Fluorouracil / Q000633:toxicity; D005544:Forecasting; D066298:In Vitro Techniques; D051379:Mice; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000187:drug effects; D018995:Myosin Heavy Chains / Q000032:analysis / Q000096:biosynthesis; D015203:Reproducibility of Results; D012518:Sarcomeres / Q000187:drug effects / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects* / Q000378:metabolism; D013723:Teratogens / Q000633:toxicity*; D014212:Tretinoin / Q000633:toxicity
D015415:Biomarkers; D013723:Teratogens; D000185:Actinin; D014212:Tretinoin; D018995:Myosin Heavy Chains; D005472:Fluorouracil