Embryonic Stem Cell Test Research Articles

The Way Forward in Reproductive/Developmental Toxicity Testing

The use of experimental animals in reproductive toxicity testing is critically reviewed on the occasion of the 50th anniversary of the publication of the Three Rs concept by Russell and Burch, since there is major concern that reproductive toxicity testing will significantly increase due to the requirements of the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system. A comparison of the test guidelines for drugs, agrochemicals and industrial chemicals shows that, for historical reasons, significantly different testing strategies are applied. The current status of development and validation of in vitro tests in reproductive toxicology is also critically evaluated. The mouse embryonic stem cell test (mEST) is the most advanced and promising of the in vitro tests. Although it has not yet been accepted for regulatory purposes, its use in preclinical drug development is well established. Moreover, promising molecular endpoints have been established in the mEST, including proteomic and toxicogenomic endpoints. Preliminary results have been obtained with a human EST (hEST). In addition, an overview is given on new in vitro reproductive toxicity tests that are currently being developed in the EU FP6 project, ReProTect, since the ReProTect test battery, which covers the essential steps of female and male fertility, implantation and embryotoxicity, holds promise for use as a screening assay for reproductive toxicity testing according to the EU REACH legislation. However, since validated in vitro methods will not be available in the short term, opportunities for the refinement of the standard in vivo tests are discussed, in order to reduce the numbers of animal used in reproductive toxicity testing. Finally, recommendations for toxicity testing in the 21st century call for the harmonisation of test methods across all areas of regulatory testing as a first step. Since the REACH system testing framework for industrial chemicals is driven by the reproductive safety testing requirements of agrochemicals, a shift is proposed to exposure-driven testing of industrial chemicals. In particular, the implementation of a new 'extended one-generation reproductive toxicity study' (EOGRTS), which includes triggers for additional testing for fertility, developmental neurotoxicity and immunotoxicity, would significantly reduce test animal numbers. It is concluded that in vitro methods hold great promise for reproductive toxicity testing in the 21st century, e.g. the ReProTect in vitro battery and the embryonic stem cell (ESC) technology focusing on molecular endpoints in both the mEST and the hEST.

Use of two validated in vitro tests to assess the embryotoxic potential of mycophenolic acid

Mycophenolate mofetil is a widely used immunosuppressive drug that recently has been categorized as a human teratogen. Animal experiments indicate a teratogenic potential of the drug, but so far, it has not been studied in embryotoxicity in vitro assays. The aim of this study was to evaluate the in vitro embryotoxic potential of mycophenolic acid and investigate the ability of such tests to detect its embryotoxic potential. We used two validated assays: the rat whole embryo culture and the murine embryonic stem cell test. Rat embryos cultured from gestational day 9.5 for 48 h with the drug showed dysmorphogenic development already at a concentration of 250 microg mycophenolic acid/l medium. At concentrations of 750 microg/l and more, all rat embryos exhibited malformations. The main effects were defective yolk sac blood circulation, neural tube defects (open cranial neural pore), malformations of the head with missing eye anlagen and heart anomalies. Moreover, the exposed embryos showed a concentration-dependent decrease in protein content, crown-rump length, number of somites and morphological score. The murine embryonic stem cell test was slightly more sensitive. Proliferation and differentiation of the ES-D3-cells were significantly impaired at concentrations of 31 and 125 microg mycophenolic acid/l medium, respectively. In the differentiation assay, at a concentration of 125 microg mycophenolic acid/l medium and more, the number of wells with differentiated cardiomyocytes significantly decreased. Additionally, a cytotoxicity assay with balb/c 3T3 mouse fibroblasts was used to compare the proliferation and vitality of embryonic cells with adult cells. In the balb/c 3T3 cytotoxicity assay, the number of vital mouse fibroblasts significantly decreased at a mycophenolic acid concentration of 62 microg/l and more. In conclusion, by using the two validated in vitro tests, we showed that mycophenolic acid exhibits a pronounced embryotoxic potential at cytotoxic concentrations. This result from validated in vitro tests is of special interest, because it supports the use of the tests to detect human teratogens.

Comparison of the Cell Differentiation Level of Mouse iPS Cells and ES-D3 Cells Using the Embryonic Stem Cell Test (EST) Protocol Attempting to Develop a New in Vitro Embryotoxicity Test

Comparison of the Cell Differentiation Level of Mouse iPS Cells and ES-D3 Cells Using the Embryonic Stem Cell Test (EST) Protocol Attempting to Develop a New in Vitro Embryotoxicity Test

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes. Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering. Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 μg/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested. Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.

Relative developmental toxicity of glycol ethers in the embryonic stem cell test and comparison to their in vivo potency ranking

Relative developmental toxicity of glycol ethers in the embryonic stem cell test and comparison to their in vivo potency ranking

A Review of the Implementation of the Embryonic Stem Cell Test (EST)

The new European chemicals policy for the Registration, Evaluation, Authorisation and Restriction of Chemicals (the REACH system), requires the routine toxicological testing of chemicals produced in volumes of more than one tonne per year, in order to enhance environmental and consumer safety (1). Under the REACH system policy, it will be most probably necessary to test several thousand chemicals for their toxicological properties.

An Attempt to Cell Recovery Factor in Cell Differentiation Culture with the Embryonic Stem Cell Test (EST)

An Attempt to Cell Recovery Factor in Cell Differentiation Culture with the Embryonic Stem Cell Test (EST)

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 Compounds

The embryonic stem cell test (EST) has been proposed as an in vitro assay that might reduce animal experimentation in regulatory developmental toxicology. So far, evaluation of the EST was not performed using compounds within distinct chemical classes. Evaluation within a distinct class of chemically related compounds can define the usefulness of the assay for the chemical class tested. The aim of the present study was to evaluate the relative sensitivity of the EST for a selected series of homologous compounds and to compare the data to the relative developmental toxicity of the compounds in vivo. To this end a series of proximate developmentally toxic glycol ether alkoxy acid metabolites was tested in the EST. All glycol ether alkoxy acid metabolites tested showed a concentration-dependent inhibition of cardiomyocyte differentiation at noncytotoxic concentrations, with methoxyacetic acid as the most potent compound followed by ethoxyacetic acid, butoxyacetic acid, and phenoxyacetic acid, respectively. The potency ranking of the compounds in the EST corresponds with the available in vivo data. The relative differences between the potencies of the compounds appeared more pronounced in the in vivo studies than in the EST. A possible explanation for this discrepancy could be the difference in the kinetics of the compounds in vivo as compared with their in vitro kinetics. This study illustrates that the EST can be used to set priorities for developmental toxicity testing within classes of related compounds.

Disentangling cellular proliferation and differentiation in the embryonic stem cell test, and its impact on the experimental protocol

The mouse embryonic stem cell test (EST) was designed to predict embryotoxicity based on the inhibition of the differentiation of embryonic stem cells (ESC) into beating cardiomyocytes in combination with cytotoxicity data in monolayer ESC cultures and 3T3 cells. In the present study, we have tested a diverse group of chemicals in the EST, applying different exposure durations, in an attempt to discriminate between effects on proliferation and differentiation within the EST protocol. Chemicals tested were monobutyl phthalate (MBP), 6-aminonicotinamide (6-AN), 5-fluorouracil (5-FU) and 5-bromo-2′-deoxyuridine (BrdU). We showed that 5-FU and BrdU behaved principally different from MBP and 6-AN. 5-FU and BrdU specifically affected cell proliferation during the first three days of the EST protocol, as shown by EB size, protein concentration and cell cycle stage analysis. In addition, we studied the differentiation state of cells in the EST protocol with time to elucidate the transition of pluripotent ESC to more differentiated cell types. Analysis by flow cytometry of the pluripotency marker SSEA-1 in EST showed that although total SSEA-1 positive cells remained unchanged up to and including day 5, the signal intensity already decreased from day 3 onwards. Furthermore, RT-PCR data showed an upregulation of the mesodermal marker T at day 3, whereas the cardiac muscle marker Myh6 was upregulated from day 5 onwards. These findings confirm that proliferation and differentiation of ESC in the EST are highly intertwined processes. Based on these findings we suggest an amended EST protocol which could more clearly discriminate between proliferation and differentiation effects of chemicals within the same EST differentiation protocol. This proposal includes a cytotoxicity assessment in EB at day 3 of the EST after day 0–3 exposure, and cardiac muscle foci counts after exposure from day 3–10 in the EST.

Embryonic Stem Cell Test Remastered: Comparison between the Validated EST and the New Molecular FACS-EST for Assessing Developmental Toxicity In Vitro

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.

Early gene expression changes during embryonic stem cell differentiation into cardiomyocytes and their modulation by monobutyl phthalate

The Embryonic Stem cell Test (EST) is an in vitro alternative test designed for the prediction of embryotoxicity. The endpoint of the test is the interference with mesoderm-derived cardiac muscle differentiation observed under the microscope as beating muscle foci. The relative subjectivity of this endpoint, as well as the applicability domain and related predictivity need further to be defined to facilitate implementation of the EST into regulatory strategies. The use of transcriptomics techniques to monitor differentiation-related gene expression changes in the EST might improve the EST in each of these aspects. Therefore, we studied the gene expression profile in embryonic stem cells (ESC) in the early phase of differentiation and its modulation by exposure to the well known embryotoxicant monobutyl phthalate (MBP). Cells were exposed from the early embryoid body stage onwards and RNA was collected after 6, 12 and 24 h of exposure. Samples were hybridized to spotted microarrays, containing 21,997-mer oligonucleotides. Differential gene expression patterns were analyzed. A total number of 43 genes that were found to be upregulated in this study as a consequence of induction of cardiomyocyte differentiation were combined in a gene set, named ‘VAN_DARTEL_HEARTDIFF_24H’. Gene Set Enrichment Analysis (GSEA) comparative analysis using multiple gene set collections clearly showed that temporal changes in gene expression were functionally related to cardiomyocyte differentiation. Furthermore, exposure of embryoid bodies (EB) to MBP increased expression of pluripotency-, proliferation- and nonmesodermal differentiation-related gene sets, which indicates inhibition of mesodermal differentiation. The inhibition of mesoderm-derived cardiomyocyte differentiation by MBP exposure was most obvious through the downregulation of our novel gene set identified in this study, ‘VAN_DARTEL_HEARTDIFF_24H’, which specifically describes the niche of early cardiomyocyte differentiation. The gene set defined in this study might serve as a starting point for defining a dedicated gene set for early detection of embryotoxicity in the EST. Such a gene set may serve as an improved endpoint in the EST as compared to morphology, and will allow a more detailed definition of the applicability domain and predictivity of EST.

The Way Forward for Reproductive/Developmental Toxicity Testing

The use of experimental animals in reproductive toxicity testing is critically reviewed on the occasion of the 50th anniversary of the publication of the Three Rs concept by Russell and Burch, since there is major concern that reproductive toxicity testing will significantly increase due to the requirements of the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system. A comparison of the test guidelines for drugs, agrochemicals and industrial chemicals shows that, for historical reasons, significantly different testing strategies are applied. The current status of development and validation of in vitro tests in reproductive toxicology is also critically evaluated. The mouse embryonic stem cell test (mEST) is the most advanced and promising of the in vitro tests. Although it has not yet been accepted for regulatory purposes, its use in preclinical drug development is well established. Moreover, promising molecular endpoints have been established in the mEST, including proteomic and toxicogenomic endpoints. Preliminary results have been obtained with a human EST (hEST). In addition, an overview is given on new in vitro reproductive toxicity tests that are currently being developed in the EU FP6 project, ReProTect, since the ReProTect test battery, which covers the essential steps of female and male fertility, implantation and embryotoxicity, holds promise for use as a screening assay for reproductive toxicity testing according to the EU REACH legislation. However, since validated in vitro methods will not be available in the short term, opportunities for the refinement of the standard in vivo tests are discussed, in order to reduce the numbers of animal used in reproductive toxicity testing. Finally, recommendations for toxicity testing in the 21st century call for the harmonisation of test methods across all areas of regulatory testing as a first step. Since the REACH system testing framework for industrial chemicals is driven by the reproductive safety testing requirements of agrochemicals, a shift is proposed to exposure-driven testing of industrial chemicals. In particular, the implementation of a new 'extended one-generation reproductive toxicity study' (EOGRTS), which includes triggers for additional testing for fertility, developmental neurotoxicity and immunotoxicity, would significantly reduce test animal numbers. It is concluded that in vitro methods hold great promise for reproductive toxicity testing in the 21st century, e.g. the ReProTect in vitro battery and the embryonic stem cell (ESC) technology focusing on molecular endpoints in both the mEST and the hEST.

Estimation of embryotoxic effect of fluoxetine using embryonic stem cell differentiation system

Aims Fluoxetine is an antidepressant drug of the selective serotonin reuptake inhibitor (SSRI) class, which is commonly prescribed to treat a wide spectrum of mood disorders including depression during pregnancy and lactation. Recent studies have proposed a possible association between an increase in major malformations and the maternal use of SSRI drugs during pregnancy. Here, we assess the effects of fluoxetine using a mouse ES cell differentiation system to clarify the possible association. Main methods Using a mouse embryonic stem (ES) cell differentiation system, we evaluated cell viability and differentiation affected by fluoxetine. Key findings Fluoxetine adversely affected cell viability and differentiation from undifferentiated ES cells to cardiomyocytes in a dose-dependent manner. The IC 50 values of fluoxetine for ES cells and NIH-3T3 fibroblasts were 1.79 μM and 4.67 μM, respectively, and the ID 50 value for ES cells was 3.79 μM. These results indicate that fluoxetine has strong toxicity evaluated by a mouse embryonic stem cell test (EST). Analysis of tissue-specific markers revealed that fluoxetine potently inhibits mesodermal development, although it promotes ectodermal differentiation in a lineage-specific manner. Significance These results using the in vitro ES cell assay system suggest a possible relationship between the teratogenicity of fluoxetine and its molecular mechanism.

Automated analysis of contractility in the embryonic stem cell test, a novel approach to assess embryotoxicity

The embryonic stem cell test (EST) is an ECVAM-validated assay to detect embryotoxicity. The output of the assay is the effect of test compounds on the differentiation of murine-derived embryonic stem cells (D3 cells), recorded by visual analysis of contracting cardiomyocyte-like cells. Incorporation of a system to assess the contractility in an automated manner is proposed, to increase the throughput in the EST independent of observer bias. The automated system is based on image recording of each well, resulting in the area (pixels) and frequency of contractility (Hz). Four test compounds were assessed for their embryotoxic potency in the 96-well version of the EST, with both manual and automated analysis: 6-Aminonicotinamide, Valproic Acid, Boric Acid, and Penicillin G. There was no statistically significant difference in the outcome of both methods in the fraction of contractility (p<0.05), resulting in the same rank-order of Relative Embryotoxic Potency (REP) values: 6-aminonicotinamide (1)>valproic acid (0.007-0.013)>Boric Acid (0.002-0.005)>Penicillin G (0.00001). The automated image recording of contractile cardiomyocyte-like cells in the EST allows for an unbiased high throughput method to assess the embryotoxic potency of test compounds, resulting in an outcome comparable to manual analysis.

Embryotoxicity hazard assessment of cadmium and arsenic compounds using embryonic stem cells

The Embryonic Stem Cell Test (EST) has been successfully validated as an in vitro method for detecting embryotoxicity, showing a good overall test accuracy of 78% [Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern. Lab. Anim. 30, 151–176]. Methylmercury was the only strong in vivo embryotoxicant falsely predicted as non-embryotoxic making the metal the most significant outlayer [Genschow, E., Spielmann, H., Scholz, G., Pohl, I., Seiler, A., Clemann, N., Bremer, S., Becker, K., 2004. Validation of the Embryonic Stem Cell Test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern. Lab. Anim. 32, 209–244]. The misclassification of methylmercury and the potential environmental exposure to developmental toxic heavy metals promoted our investigation of whether the EST applicability domain covers cadmium and arsenic compounds. The EST misclassified cadmium, arsenite and arsenate compounds as non-embryotoxic, even when including arsenic metabolites (methylarsonate, methylarsonous and dimethylarsinic). The reasons were the lack of higher cytotoxicity towards embryonic stem cells as compared to more mature cells (3T3 fibroblasts) or the absence of inhibition of cardiac differentiation by specific mechanisms rather than general cytotoxicity. Including EST data on heavy metals from the literature (lithium, methylmercury, trivalent chromium and hexavalent chromium) revealed that the test correctly predicted the embryotoxic potential of three out of the seven heavy metals, indicating an insufficient predictivity for such metals. Refinement of the EST prediction model and inclusion of additional toxicological endpoints could expand the applicability domain and enhance the predictive power of the test.

Optimisation of the cell cultivation methods in the embryonic stem cell test results in an increased differentiation potential of the cells into strong beating myocard cells

In order to support drug research in the selection process for non-embryotoxic pharmaceutical compounds, a screening method for embryotoxicity is needed. The murine embryonic stem cell test (EST) is a validated in vitro test based on two permanent mouse cell lines and delivering results in 10-days. Implementation of this test within our laboratory, revealed variability in the differentiation potential of the embryonic stem cells and, as a consequence, a lot of assays needed to be rejected due the fact the acceptance criteria were not reached. In order to gain a better yield of contracting myocardial cells, we used (1) a stringent control of the cell growth during subcultivation and a standardised hanging drop culture method and (2) a non-enzymatic cell harvest instead of a trypsin/EDTA cell harvest. Implementing of these cell culture modifications resulted in a decreased variability in the size of embryonic bodies, an increase of the number of acceptable tests and a significant increase of the differentiation potential of embryonic cells into strong beating myocardium, which made scoring less time consuming. Testing of 6 reference compounds in the optimized EST showed that the cell culture modifications did not changed the in vitro classification.

Evaluation of the Embryotoxic Potency of Compounds in a Newly Revised High Throughput Embryonic Stem Cell Test

The ability of murine-derived embryonic stem cells (D3) to differentiate into cardiomyocytes is the basis of the embryonic stem cell test (EST). With the EST, chemicals and pharmaceuticals can be assessed for their embryotoxic potency early on in the development process. In order to come to a higher throughput EST, a 96-well based method was developed based on low attachment well plates that allow for the formation of embryonic bodies from which the stem cells can differentiate. Twelve test compounds were selected based on their reported in vitro and in vivo embryotoxic potency. In the 96-well based EST, reportedly strong embryotoxic compounds 5-fluorouracil, 6-aminonicotinamide (6AN), methylmercury chloride, and hydroxyurea were correctly ranked with corresponding Relative Embryotoxic Potency values (REP, based on the EC(50) (microM) value of 6AN) of 2.6 +/- 2.9, 1, 2.0 +/- 3.1, and 0.07 +/- 0.05, respectively. Moderately embryotoxic compounds valproic acid, boric acid, methoxyacetic acid, and lithium chloride resulted in a correct ranking with REP values of 0.01 +/- 0.003, 0.001 +/- 0.001, 0.0007 +/- 0.001, and 0.0006 +/- 0.0004, respectively. The included nonembryotoxic compounds Penicillin G, acrylamide, and saccharin did not result in an inhibition of D3 cells to differentiate into cardiomyocytes, other than related to cytotoxicity (REP value of 0.00001). However, diphenhydramine resulted in an inhibitory effect similarly to the strong embryotoxic compound hydroxyurea, with a REP value of 0.40 +/- 0.36. However, further evaluation suggested this was due to direct inhibition of the contractile capacity of the D3 cardiomyocytes, rather than an embryotoxic mechanism. The 96-well based EST is a promising addition to the screening process of newly developed chemicals and pharmaceuticals.

Assessment of the Embryonic Stem Cell Test and application and use in the pharmaceutical industry

The European Centre for the Validation of Alternative Methods (ECVAM) designed the Embryonic Stem Cell Test (EST) as a tool for classifying developmentally toxic compounds. An in vitro tool to assess developmental toxicity would be of great value to the pharmaceutical industry to help with toxicity-associated attrition. ECVAM's EST protocol was used, but employing a different mouse embryonic stem cell (ESC) line and an alternative differentiation medium. A subset of the compounds used to validate the EST assay along with a number of in-house pharmaceutical compounds plus marketed pharmaceutical compounds were used to assess the EST performance with receptor-mediated compounds. Our results with ECVAM compounds mirrored ECVAM's. Compounds that were developmentally toxic in vivo were classified by the EST as moderate risk. Overall, the accuracy was 75% with the current set of data and the predictivity of low-, moderate-, and high-risk compounds was 90, 71, and 60% while the precision was 59, 86, and 100%, respectively. Interestingly, a number of the non-developmentally toxic compounds had values for the 3T3 IC(50) values, which were lower than the ESC IC(50) and ID(50), a situation not taken into account by ECVAM when designing the EST algorithm. The assay as currently constructed has a significant false-positive rate (approximately 40%), but a very low false-negative rate (approximately 7%). Additional moderate- and high-risk compounds need to be assessed to increase confidence, accuracy, and understanding in the EST's predictivity.

Embryotoxicity hazard assessment of methylmercury and chromium using embryonic stem cells

The embryonic stem cell test (EST) has been scientifically validated (2001) as an in vitro embryotoxicity test, showing a good overall test accuracy of 78%. Methylmercury (MeHg) was the most significant outlayer identified, as the metal was the only strong in vivo embryotoxicant falsely predicted to be non-embryotoxic. The EST misclassification of MeHg, and the potential environmental exposure and developmental toxic hazards of heavy metals gave us the rationale to investigate whether the EST can correctly predict the embryotoxic potential of two heavy metals different from MeHg. The EST correctly classified trivalent chromium to be non-embryotoxic and hexavalent chromium to be embryotoxic, while we confirmed the misclassification of MeHg. MeHg causes developmental abnormalities in the brain. We therefore aimed to improve the in vitro prediction of MeHg embryotoxicity by including a neuronal ES cell differentiation assay. Differentiation of neuronal-like cells was demonstrated by real-time PCR experiments, showing up-regulation of several neuronal marker genes, and immunohistochemistry, demonstrating the appearance of nestin, neurofilament medium polypeptide, β-tubulin III and microtubule-associated protein 2 (Mtap2) positive cells. We identified Mtap2 mRNA expression as a sensitive toxicological endpoint for MeHg-induced neuronal embryotoxicity, as Mtap2 mRNA was down-regulated in the presence of non-cytotoxic concentrations of MeHg. Noticeably, several other neuronal marker genes were unaffected by MeHg and Mtap2 expression was not affected until day 14 of differentiation. This implies that the total neuronal-like cell number was unchanged and that the down-regulation of Mtap2 expression reflects neuron-specific toxicity, i.e. instability of the neuron-specific microtubules, and arrest of the neuronal maturation. The fact, that most marker genes were unaffected by MeHg, stresses the importance of including an array of marker genes. In conclusion, our results imply that inclusion of additional target tissues and refinement of the current prediction model may enhance the predictive power of the EST.

First steps in establishing a developmental toxicity test method based on human embryonic stem cells

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.