Mouse embryonic stem cells (mESCs) represent an attractive cellular system for in vitro studies in developmental biology as well as toxicology because of their potential to differentiate into all fetal cell lineages. The present study aims to establish an in vitro system for developmental neurotoxicity testing employing mESCs. We developed a robust and reproducible protocol for fast and efficient differentiation of the mESC line D3 into neural cells, optimized with regard to chemical testing. Morphological examination and immunocytochemical staining confirmed the presence of different neural cell types, including neural progenitors, neurons, astrocytes, oligodendrocytes, and radial glial cells. Neurons derived from D3 cells expressed the synaptic proteins PSD95 and synaptophysin, and the neurotransmitters serotonin and γ-aminobutyric acid. Calcium ion imaging revealed the presence of functionally active glutamate and dopamine receptors. In addition, flow cytometry analysis of the neuron-specific marker protein MAP2 on day 12 after induction of differentiation demonstrated a concentration dependent effect of the neurodevelopmental toxicants methylmercury chloride, chlorpyrifos, and lead acetate on neuronal differentiation. The current study shows that D3 mESCs differentiate efficiently into neural cells involving a neurosphere-like state and that this system is suitable to detect adverse effects of neurodevelopmental toxicants. Therefore, we propose that the protocol for differentiation of mESCs into neural cells described here could constitute one component of an in vitro testing strategy for developmental neurotoxicity.

Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition.

The embryonic stem cell test (EST) represents a reliable, scientifically validated in vitro system for the detection and classification of compounds according to their teratogenic potency. However, some serious issues were frequently raised against the widespread implementation and practicability of the EST in its original version. Most importantly, the evaluation of the morphological endpoint of beating cell agglomerates requires extensive experimental experience and is prone to misjudgment. Also, the testing period of 10 days is too long and costly to be attractive for industries interested in high-throughput screening of potential drug candidates. These drawbacks prompted us to work out a new molecular approach based on analysis of the expression of certain marker proteins specific for developing heart tissue. We have previously reported that quantitative flow cytometry of marker proteins (i.e., sarcomeric myosin heavy chain and alpha-actinin) can be performed at day 7 in embryonic stem cells from mice and combined with concurrent cell viability analysis. In the present study, extensive investigations were performed in order to explore the predictive power and validity of the newly established EST, subsequently referred to as molecular fluorescence activated cell sorting (FACS)-EST, by applying and comparing a set of 10 well-known embryotoxicants that encompasses the full range of chemical inherent embryotoxic potencies possible. While the molecular FACS-EST offered the same sensitivity compared to the validated EST protocol, the test duration could be significantly reduced. Due to significant improvements, this new molecular method holds promise as a sensitive, more rapid and reproducible screen highly suited to predict developmental toxicity in vivo from in vitro data.

In the field of reproductive toxicity mandatory test guidelines require in vivo experiments for the detection of the embryotoxic potential of chemicals and drugs. A promising alternative method for these purposes has been provided with the Embryonic Stem Cell Test (EST). This assay is based on the capacity of murine embryonic stem cells (ES cells) to differentiate in vitro into a variety of cell types. The EST is able to assess the embryotoxic potential of chemicals by the evaluation of inhibitory effects on differentiation of contracting myocardial cells which can be detected by microscopical analysis. Using a biostatistical prediction model (PM) the assay passed an international validation study and was able to predict the embryotoxic potential of test chemicals with an accuracy of 78%.A joint project was carried out by ZEBET and German pharmaceutical companies to improve the EST by establishing molecular endpoints of differentiation. Cardiac-specific gene expression has been studied at protein and RNA levels by flow cytometry and real-time-PCR under the influence of 10 chemicals with different embryotoxic potentials. The results obtained using cardiac-specific molecular endpoints were comparable to the validated microscopic analysis of beating areas and led to the same predictive outcome. The data clearly demonstrated that the selected molecular markers provide objective endpoints of early embryonic differentiation and are able to predict developmental toxicity in vivo from in vitro data for reference compounds.In conclusion, a modified EST holds promise to be a new predictive screening system for hazard assessment with regard to developmental toxicity.The first two authors contribute equally.

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 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 outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts. Through a number of prevalidation and validation studies, the EST has been demonstrated to be a reliable alternative method for embryotoxicity testing based on the most important mechanisms in embryotoxicity-cytotoxicity and differentiation--as well as on differences in sensitivity between differentiated and embryonic tissues. Improvements of the EST protocol using flow cytometry analysis showed that differential expression of sarcomeric myosin heavy chain and alpha-actinin proteins quantified under the influence of a test compound is a useful marker for detecting potential teratogenicity. The in vitro embryotoxicity test described in this chapter is rapid, simple, and sensitive and can be usefully employed as a component of the risk/hazard assessment process.

ALTEX 22, Special Issue 2005 278 Living organisms from single cells to people can be thought of as “biological machines” – feedback control systems following genetically-determined developmental programs and in adulthood focusing on homeostasis and reproduction. Systems engineering principles that define the control circuits in man-made machines are also applicable to living systems. In fact, striking parallels exist between control circuits in complex machines and in biological cells and tissues (Carlson and Doyle, PNAS 99, Suppl. 1, 2538-2545, 2002). Regulatory networks exist at all levels of biological organisation – molecular, cellular, tissue and organism – and a systems engineering approach to characterising their structure and function appears to be possible. We can ask if and when computational models will be ready to replace laboratory animals in toxicological research and testing. First, however, we should recall a cardinal rule of computer programming – garbage in – garbage out. In other words, a robust, predictive computational model of a biological system must be based on a sound understanding of that system. The rate-limiting step in the development of these models is the rate of our progress in understanding the relevant biology. Although a revolution is underway in the study of basic biology it will be some time before we can draw inclusive circuit diagrams of living cells and tissues. Today’s computational models are thus incomplete and are not suitable replacements for laboratory animals. Computational models do, however, have important roles to play as adjuncts to classical toxicological methods. Three dimensional modelling of protein structure, for example, can be used to screen chemical structures for binding behaviours potentially associated with toxic effects. Physiologically-based pharmacokinetic models help to ensure efficient experimental design and thereby refine animal use. The ongoing, rapid development of new biological understanding and the explosive growth of computer hardware and software technologies guarantee that the role of computational modelling in toxicology will expand continually. While these developments will not, in the foreseeable future, eliminate the need for laboratory animals, they will lead to significant refinement and possibly to reduction of animal use. Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy. Lecture Computational modelling of biological systems: Implications for use of laboratory animals in toxicological testing and research

The embryonic stem cell test (EST) is an in vitro assay that has been developed to assess the teratogenic and embryotoxic potential of drugs and chemicals. It is based on the capacity of murine ES cells (cell line D3) to differentiate into contracting myocardial cells under specific cell culture conditions. The appearance of beating cardiomyocytes in embryoid body (EB) outgrowths is used as a toxicological endpoint to assess the embryotoxic potential of a test substance. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study the EST predicted the embryotoxic potential of chemicals and drugs with the same reliability as two other in vitro embryotoxicity tests, which employed embryonic cells and tissues from pregnant animals. In a joint research project with German pharmaceutical companies we have successfully improved the EST by establishing molecular endpoints of differentiation in cultured ES cells. The quantification of cardiac-specific protein expression by intracellular flow cytometry has been studied in the presence of chemicals of different embryotoxic potential. The results obtained using molecular endpoints specific for differentiated cardiomyocytes employing FACS (fluorescence-activated cell sorting) analysis will be presented in comparison to the validated endpoint - the microscopic analysis of beating areas. FACS analysis provides a more objective endpoint for predicting the embryotoxic potential of chemicals than the validated method. Furthermore, flow cytometry promises to be suitable for high-throughput screening systems (HTS). In addition, our partners from the joint project have improved the EST by developing protocols that stimulate differentiation of ES cells into neural and endothelial cells, chondrocytes and osteoblasts, because some substances might have embryotoxic effects on specific cell-types other than cardiomyocytes. These protocols have been successfully established at ZEBET and in the participating laboratories. Additionally, molecular endpoints have been established for the detection of specific differentiation pathways. Furthermore, new prediction models (PMs) have been developed using single endpoints of the EST.

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 α-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 α-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, α-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.

Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse can be induced to differentiate in culture into a variety of cell types, including cardiac muscle cells. In the embryonic stem cell test (EST) the capacity of ES cells of the mouse cell line D3 to differentiate into contracting cardiomyocytes is used to assess the embryotoxic potential of test compounds and in addition, the effects on the viability of ES cells and differentiated mouse fibroblasts (cell line 3T3) are compared. The three endpoints are used to classify the embryotoxic potential of chemicals after 10 days of exposure: (i) the inhibition of differentiation of ES cells into cardiomyocytes (ID50) and (ii) the decrease of viability of 3T3 cells (IC503T3) and (iii) ES cells (IC50D3) in a MTT cytotoxicity test. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study funded by ECVAM it could be demonstrated that the EST can predict the embryotoxic potential of a test compound as good as frequently used mammalian systems based on pregnant animals. In a joint project with major German pharmaceutical companies we are attempting to improve the EST by establishing molecular endpoints of differentiation (e.g. cardiac, neuronal, chondrogenic) in cultured ES cells. We have studied the expression of tissue specific proteins in ES cell cultures in the presence of embryotoxic chemicals by immunofluorescent antibody techniques, e.g. FACS analysis. The other groups are focusing on endogenous gene expression in early development by RT-PCR methods or the DNA microarray technique. The results obtained recently using molecular markers specific for cardiac differentiation and employing intracellular flow cytometry for quantification will be presented. Molecular endpoints will allow improvement of the EST by measuring gene expression patterns in a small number of murine ES cells.

The embryonic stem cell test (EST) which is based on the morphometric measurementof cardiomyocyte differentiation and cytotoxicity parameters represents a validatedmethod for the detection and classification of compounds according to theirteratogenic potency. However, more work is required to assess its applicability domainand to improve its predictive capacity before gaining regulatory acceptance.We picked valproic acid (VPA) as a model compound to evaluate the suitability ofthe EST for distinguishing between developmental toxicity potencies of substanceswith closely related structures. Here we investigated six closely related analogues ofVPA whose teratogenic potential has been previously determined in the NMRI exencephalymouse model. Determining the concentration of VPA and of each of itssix derivatives at which half maximal inhibition of differentiation occurs in the ESTrevealed a similar ranking as found previously. Distinct embryotoxicities in vivo ofstereoisomers which differ only in their spatial configuration were reproduced bythe EST. Similarly, an increased potency in vivo correlating with longer chain lengthof the congener was evident as higher toxicity in the EST. Our data demonstratethat the EST is capable of differentiating among closely related molecules accordingto their embryotoxic potency. As toxicological endpoints, both differentiation andcytotoxicity in vitro have to be considered to assess teratogenicity comparable to invivo results. In conclusion, all substances were ranked in the same order and in fullaccordance to data obtained in the NMRI exencephaly mouse model.

The embryonic stem cell test (EST) is an in vitro assay that has been developed to assess the teratogenic and embryotoxic potential of drugs and chemicals. It is based on the capacity of murine ES cells (cell line D3) to differentiate into contracting myocardial cells under specific cell culture conditions. The appearance of beating cardiomyocytes in embryoid body (EB) outgrowths is used as a toxicological endpoint to assess the embryotoxic potential of a test substance. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study the EST predicted the embryotoxic potential of chemicals and drugs with the same reliability as two other in vitro embryotoxicity tests, which employed embryonic cells and tissues from pregnant animals. In a joint research project with German pharmaceutical companies we have successfully improved the EST by establishing molecular endpoints of differentiation in cultured ES cells. The quantification of cardiac-specific protein expression by intracellular flow cytometry has been studied in the presence of chemicals of different embryotoxic potential. The results obtained using molecular endpoints specific for differentiated cardiomyocytes employing FACS (fluorescence-activated cell sorting) analysis will be presented in comparison to the validated endpoint-the microscopic analysis of beating areas. FACS analysis provides a more objective endpoint for predicting the embryotoxic potential of chemicals than the validated method. Furthermore, flow cytometry promises to be suitable for high-throughput screening systems (HTS). In addition, our partners from the joint project have improved the EST by developing protocols that stimulate differentiation of ES cells into neural and endothelial cells, chondrocytes and osteoblasts, because some substances might have embryotoxic effects on specific cell-types other than cardiomyocytes. These protocols have been successfully established at ZEBET and in the participating laboratories. A d d i t i o n a l l y, molecular endpoints have been established for the detection of specific differentiation pathways. Furthermore, new prediction models (PMs) have been developed using single endpoints of the EST. This manuscript is an invited paper and contains the results presented in three posters at the Linz 2003 meeting. The authors were asked to discuss the trends in improving the EST further.