In the embryonic stem cell test (EST), differentiation of mouse embryonic stem cells (mESCs) is used as a model to assess embryotoxicity in vitro. The test was successfully validated by the European Center for the Validation of Alternative Methods (ECVAM) and models fundamental mechanisms in embryotoxicity, such as cytotoxicity and differentiation. In addition, differences in sensitivity between differentiated (adult) and embryonic cells are also taken into consideration. To predict the embryotoxic potential of a test substance, three endpoints are assessed: the inhibition of differentiation into beating cardiomyocytes, the cytotoxic effects on stem cells and the cytotoxic effects on 3T3 fibroblasts. A special feature of the EST is that it is solely based on permanent cell lines so that primary embryonic cells and tissues from pregnant animals are not needed. In this protocol, we describe the ECVAM-validated method, in which the morphological assessment of contracting cardiomyocytes is used as an endpoint for differentiation, and the molecular-based FACS-EST method, in which highly predictive protein markers specific for developing heart tissue were selected. With these methods, the embryotoxic potency of a compound can be assessed in vitro within 10 or 7 d, respectively.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Schaefer, C., Peters, P.W.J. & Miller, R.K. Drugs During Pregnancy and Lactation (Elsevier, Academic Press, 2007).
International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). ICH harmonized tripartite guideline: detection of toxicity to human reproduction for medicinal products & toxicity to male fertility (S5 (R2)). Current Step 4 version. (1993). http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S5_R2/Step4/S5_R2__Guideline.pdf.
Spielmann, H., Pohl, I., Döring, B., Liebsch, M. & Moldenhauer, F. The embryonic stem cell test (EST), an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In Vitro Toxicol. 10, 119–127 (1997).
Hoffman, J.A. & Merrill, B.J. New and renewed perspectives on embryonic stem cell pluripotency. Front. Biosci. 12, 3321–3332 (2007).
Keller, G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 19, 1129–1155 (2005).
Wobus, A.M. & Boheler, K. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol. Rev. 85, 635–678 (2005).
Laschinski, G., Vogel, R. & Spielmann, H. Cytotoxity test using blastocyst-derived euploid embryonal stem cells: a new approach to in vitro teratogenesis screening. Reprod. Toxicol. 5, 57–64 (1991).
Smith, A.G. Embryo-derived stem cells: of mice and men. Annu. Rev. Cell. Dev. Biol. 17, 435–462 (2001).
Wobus, A.M., Guan, K., Yang, H.T. & Boheler, K.R. Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation. Methods Mol. Biol. 185, 127–156 (2002).
Doetschmann, T., Eistetter, H.R., Schmidt, W. & Kemler, R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87, 7–45 (1985).
Martin, G.R., Wiley, L.M. & Damjanov, I. The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells. Dev. Biol. 61, 220–244 (1977).
Genschow, E. et al. Development of prediction models for three in vitro embryotoxicity tests. In vitro Mol. Toxicol. 13, 51–65 (2000).
Genschow, E. et al. The ECVAM international validation study on in vitro embrytoxicity tests. Results of the definitive phase and evaluation of prediction models. Altern. Lab. Anim. 30, 151–176 (2002).
Genschow, E. et al. Validation of the embryonic stem cell test (EST) in the ECVAM international validation study on in vitro embryotoxicity. Altern. Lab. Anim. 32, 209–244 (2004).
Piersma, A.H. INVITTOX protocol no. 123: embryotoxicity testing in post-implantation embryo culture-method of piersma. http://ecvam-dbalm.jrc.ec.europa.eu/public_view_doc2.cfm?id=15BFE9AC6ECC8ED3D7F6DF03B5D5BBE97180BB0BC12CB10496CDA74B54630A05A3291B895581F634(updated 2010).
Flint, O.P. & Orton, T.C. An in vitro assay for teratogens with cultures of rat embryo mid-brain and limb cells. Toxicol. Appl. Pharmacol. 76, 383–395 (1984).
Bigot, K., De Lange, J., Archer, G., Clothier, R. & Bremer, S. The relative semi-quantification of mRNA expression as a useful toxicological endpoint for the identification of embryotoxic/teratogenic substances. Toxicol. In Vitro 13, 619–623 (1999).
Pellizzer, C., Adler, S., Corvi, R., Hartung, T. & Bremer, S. Monitoring of teratogenic effects in vitro by analysing a selected gene expression pattern. Toxicol. In Vitro 18, 325–335 (2004).
Chapin, R.E. & Stedman, D.B. Endless possibilities: stem cells and the vision for toxicology testing in the 21st century. Toxicol. Sci. 105, 342–350 (2008).
van Dartel, D.A.M. et al. Evaluation of developmental toxicant identification using gene expression profiling in embryonic stem cell differentiation cultures. Toxicol. Sci. 119, 126–134 (2011).
Osman, A.M. et al. Proteome profiling of mouse embryonic stem cells to define markers for cell differentiation and embryotoxicity. Reprod. Toxicol. 30, 322–332 (2010).
Groebe, K. et al. Protein biomarkers for in vitro testing of embryotoxicity. J. Proteome Res. 9, 5727–5738 (2010).
Peters, K.A. et al. Evaluation of the embryotoxic potency of compounds in a newly revised high throughput embryonic stem cell test. Toxicol. Sci. 105, 342–350 (2008).
Peters, K.A. et al. Automated analysis of contractility in the embryonic stem cell test, a novel approach to assess embryotoxicity. Toxicol. In Vitro 22, 1948–1956 (2008).
De Smedt, A. et al. Optimization of the cell cultivation methods in the embryonic stem cell test results in an increased differentiation potential of the cell into strong beating myocard cells. Toxicol. In Vitro 22, 1789–1796 (2008).
van Dartel, D.A.M., Zeijen, N.J.L., de la Fonteyne, L.J.J., van Schooten, F.J. & Piersma, A.H. Disentangling cellular proliferation and differentiation in the embryonic stem cell test, and its impact on the experimental protocol. Reprod. Toxicol. 28, 254–261 (2009).
Spielmann, H. et al. The practical application of the three validated in vitro embryotoxicity tests. Altern. Lab. Anim. 34, 527–538 (2006).
Marx-Stoelting, P. et al. A review of the implementation of the embryonic stem cell test (EST). Altern. Lab. Anim. 37, 313–328 (2009).
Seiler, A., Visan, A., Buesen, R., Genschow, E. & Spielmann, H. Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test. Reprod. Toxicol. 18, 231–240 (2004).
Buesen, R. et al. Embryonic stem cell test (EST) remastered: comparison between the validated EST and the new molecular FACS-EST for assessing developmental toxicity in vitro. Toxicol. Sci. 108, 389–400 (2009).
Maltsev, V.A., Wobus, A.M., Rohwedel, J., Bader, M. & Hescheler, J. Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ. Res. 75, 233–244 (1994).
Murry, C.E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008).
Sachinidis, A. et al. Cardiac specific differentiation of mouse embryonic stem cells. Cardiovasc. Res. 58, 278–291 (2003).
Boheler, K.R. et al. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ. Res. 91, 189–201 (2002).
Scholz, G. & Spielmann, H. INVITTOX protocol 113. Embryonic Stem Cell Test (EST). http://ecvam-dbalm.jrc.ec.europa.eu/public_view_doc2.cfm?id=DC5ABDF7AC30F1B7DF7EF27E87D68AAC7180BB0BC12CB10496CDA74B54630A05A3291B895581F634 (updated 2010).
Spielmann, H. Predicting the risk of developmental toxicity from in vitro assays. Toxicol. Appl. Pharmacol. 207, 375–380 (2005).
Piersma, A.H. Alternative methods for developmental toxicity testing. Basic Clin. Pharm. Toxicol. 98, 427–431 (2006).
Brown, N.A. et al. Screening chemicals for reproductive toxicity: the current alternatives. The report and recommendations of an ECVAM/ETS workshop (ECVAM workshop 12). Altern. Lab. Anim. 23, 868–882 (1995).
Brown, N.A. INVITTOX protocol no. 122: The Micromass Test—Method of Brown. http://ecvam-dbalm.jrc.ec.europa.eu/public_view_doc2.cfm?id=7DDBC8A6A03A1DB626729D0A17A0D2FF7180BB0BC12CB10496CDA74B54630A05A3291B895581F634 (updated 2010).
Spielmann, H. et al. Validation of the micromass assay (MM) in the ECVAM international validation study on in vitro embryotoxicity. Altern. Lab. Anim. 32, 245–274 (2004).
Piersma, A.H. et al. Validation of the rat postimplantation whole embryo culture in the ECVAM international validation study on in vitro embryotoxicity. Altern. Lab. Anim. 32, 275–307 (2004).
Balls, M. & Hellsten, E. Statement of the scientific validity of the embryonic stem cell test (EST)—an in vitro test for embryotoxicity. Statement of the scientific validity of the micromass test—an in vitro test for embryotoxicity. Statement of the scientific validity of the postimplantation rat whole embryo culture assay—an in vitro test for embryotoxicity. Altern. Lab. Anim. 30, 265–273 (2002).
Stummann, T.C., Hareng, L. & Bremer, S. Embryotoxicity hazard assessment of cadmium and arsenic compounds using embryonic stem cells. Toxicol 252, 118–122 (2008).
Schenk, B. et al. The ReProTect feasibility study, a novel comprehensive in vitro approach to detect reproductive toxicants. Reprod. Toxicol. 30, 200–218 (2010).
OEHHA. Evidence on Developmental and Reproductive Toxicity of Inorganic Arsenic. Office of Environmental Health Hazard Assessment, 68. http://www.oehha.ca.gov/prop65/pdf/AS-HID.pdf (1996).
Brown, N.A. Selection of test chemicals for the ECVAM international validation study on in vitro embryotoxicity tests. Altern. Lab. Anim. 30, 177–198 (2002).
International Program on Chemical Safety (IPCS, INCHEM). WHO/FAO data sheets on pesticides. No. 84, Nitrofen. http://www.inchem.org/documents/pds/pds/pest84_e.htm. (1996).
Paquette, J.A. et al. Assessment of the embryonic stem cell test and application and use in the pharmaceutical industry. Birth Defects Res. B Dev. Reprod. Toxicol. 283, 104–111 (2008).
De Jong, E. et al. Relative developmental toxicity of glycol ether alkoxy acid metabolites in the embryonic stem cell test as compared with the in vivo potency of their parent compound. Toxicol. Sci. 110, 117–124 (2009).
Riebeling, C. et al. Evaluation of structure-activity relationships in the teratogenicity of valproic acid derivatives using the embryonic stem cell test. Toxicol. Sci. 120, 360–370 (2011).
West, P.R., Weir, A.M., Smith, A.M., Donley, E.L.R. & Cezar, G.G. Predicting human developmental toxicity of pharmaceuticals using human embryonic stem cells and metabolomics. Toxicol. Appl. Pharmacol. 247, 18–27 (2009).
Wobus, A.M., Wallukat, G. & Hescheler, J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 48, 173–182 (1991).
Bader, D., Masaki, T. & Fischmann, D.A. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J. Cell. Biol. 95, 763–770 (1982).
Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. lmmunol. Methods 65, 55–63 (1983).
BD CellQuest Pro software user's guide (Becton Dickinson, Heidelberg, Germany). http://facs.stanford.edu/sff/doc/cellquestprouserguide.pdf (2002).
Shapiro, H.M. Measuring cell surface and intracellular antigens. In Practical Flow Cytometry edn. 4 (ed. Shapiro, H.M.) 345–361 (John Wiley & Sons, 2003).
Ritz, C. & Streibig, J.C. Bioassay analysis using R. J. Statist. Software 12, 1–22 (2005).
Scholz, G. et al. Prevalidation of the embryonic stem cell test (EST)—A new in vitro embryotoxicity test. Toxicol. In Vitro 13, 675–681 (1999).
Eppenberger-Eberhardt, M., Flamme, I., Kurer, V. & Eppenberger, H.M. Reexpression of alpha-smooth muscle actin isoform in cultured adult rat cardiomyocytes. Dev. Biol. 139, 269–278 (1990).
We thank A. Visan and R. Buesen for conducting flow cytometry analyses, EST experiments and preparing illustrations, B. Slawik for excellent technical assistance, R. Pirow for excellent statistical support, C. Riebeling for critical reading of the manuscript and for his valuable contribution to the preparation of the figures and G. Friedmann-Marohn for his excellent technical support in preparing high-resolution artwork.
This work was supported in part by the German Federal Ministry of Education and Research BMBF grants 0312312 and 0313070A.
The authors declare no competing financial interests.
About this article
Cite this article
Seiler, A., Spielmann, H. The validated embryonic stem cell test to predict embryotoxicity in vitro. Nat Protoc 6, 961–978 (2011). https://doi.org/10.1038/nprot.2011.348
An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds
Cell Biology and Toxicology (2021)
Combination of the BeWo b30 placental transport model and the embryonic stem cell test to assess the potential developmental toxicity of silver nanoparticles
Particle and Fibre Toxicology (2020)
Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4
Scientific Reports (2020)
Archives of Toxicology (2020)
Chromosomal instability reducing effect of paclitaxel and lapatinib in mouse embryonic stem cells with chromosomal abnormality
Molecular Biology Reports (2020)