Embryonic Stem Cell Test Research Articles

Advanced developmental toxicity test method based on embryoid body’s area

Embryonic stem cell test (EST) evaluates the embryotoxic potential of substances and measures the half inhibition in viability of mouse embryonic stem cells (ESCs), fibroblasts (3T3 cells) and in cardiac differentiation of ESC. In this study, we suggest the developmental toxicity test method (termed EBT) applying area of embryoid bodies (EBs) instead of cardiac differentiation of EST. In the assessment of 21 substances, EB area was logarithmically decreased in dose-dependent manner. Decline in EB area resulted in decrease of beating ratio during differentiation of ESCs. In classification by the EBT-based prediction model reflecting decline in cell viability and EB area, toxicity for 21 chemicals showed 90.5% accuracy. In the results of next generation sequencing, reduction in EB area resulted from cell cycle arrest mediated by HDAC2 and CDKN2A. Conclusively, EBT is advanced and is a useful tool to assess and classify various embryotoxicants in a short time with less effort.

Editor's Highlight: Development of Novel Neural Embryonic Stem CellTests for High-Throughput Screening of Embryotoxic Chemicals.

There is a great demand for appropriate alternative methods to rapidly evaluate the developmental and reproductive toxicity of a wide variety of chemicals. We used the differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes as a basis for establishing a rapid and highly reproducible invitro embryotoxicity test known as the Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST). In this study, we developed novel neural-Luc ESTs using two marker genes for neural development, tubulin beta-3 (Tubb3) and Reelin (Reln), and evaluated the capacity of these tests to predict developmental toxicity. In addition, we tested whether an integrated approach (a combination of neural-Luc ESTs and the Hand1-Luc EST) improved developmental toxicant detection. To perform our neural-Luc ESTs, we needed to generate stable transgenic mESCs with individual promoters linked to the luciferase gene, and to establish that similar changes in promoter activities and mRNA expression levels occur during neural differentiation. Based on the concentration-response curves of 15 developmental toxicants and 17 non-developmental toxic chemicals, we derived a prediction formula and assessed the capacity of this formula to predict developmental toxicity. Although both were highly sensitive and specific for predicting developmental toxicity, neural-Luc ESTs had similar predictive capacities. In contrast, neural-Luc ESTs and Hand1-Luc EST had significantly different predictive powers. As expected, the combination of these ESTs increased the sensitivity of developmental toxicant detection. These results demonstrate the convenience and the usefulness of this combination of ESTs as an alternative assay system for future toxicological and mechanistic studies of developmental toxicity.

Interactions between three typical endocrine-disrupting chemicals (EDCs) in binary mixtures exposure on myocardial differentiation of mouse embryonic stem cell

In recent years, various kinds of endocrine-disrupting chemicals (EDCs) have been detected in human blood and urine. Thus, it was important to investigate the combined toxicity effect of EDCs. In the present study, we evaluated the individual and combined developmental toxicities of three classic EDCs: perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and bisphenol A (BPA) by embryonic stem cell test (EST). The similarities and differences between combination of same chemical group as well as different chemical groups were investigated in this research. Our results showed that the three compounds were all classified as weak embryotoxicity. The results of co-exposure revealed that there was synergistic action in combination of PFOS and BPA on myocardial differentiation. However, in all endpoints, the combined effects between PFOA with PFOS or BPA were both additive action. Therefore, we concluded that the additive effects were found in most different EDC mixtures whether they had similar structure or not. On the other hand, synergistic action was observed in a mixture of EDCs that belonged to a different chemical groups.

Prediction of Embryotoxicity by Four Kinds of Fluorine Compounds Based on Embryonic Stem Cell Test (EST) Protocol

Prediction of Embryotoxicity by Four Kinds of Fluorine Compounds Based on Embryonic Stem Cell Test (EST) Protocol

Transcriptomics analysis of early embryonic stem cell differentiation under osteoblast culture conditions: Applications for detection of developmental toxicity

The mouse embryonic stem cell test (mEST) is a promising in vitro assay for predicting developmental toxicity. In the current study, early differentiation of D3 mouse embryonic stem cells (mESCs) under osteoblast culture conditions and embryotoxicity of cadmium sulfate were examined. D3 mESCs were exposed to cadmium sulfate for 24, 48 or 72h, and whole genome transcriptional profiles were determined. The results indicate a track of differentiation was identified as mESCs differentiate. Biological processes that were associated with differentiation related genes included embryonic development and, specifically, skeletal system development. Cadmium sulfate inhibited mESC differentiation at all three time points. Functional pathway analysis indicated biological pathways affected included those related to skeletal development, renal and reproductive function. In summary, our results suggest that transcriptional profiles are a sensitive indicator of early mESC differentiation. Transcriptomics may improve the predictivity of the mEST by suggesting possible modes of action for tested chemicals.

Embryotoxicity estimation of commonly used compounds with embryonic stem cell test.

The embryonic stem cell test (EST), an alternative model to animal studies, is a reliable and scientifically validated in vitro system for testing embryotoxicity. In contrast to most in vivo animal tests, two permanent cell lines, murine fibroblasts (BALB/c-3T3 cells) and murine embryonic stem cells (mES-D3 cells), are used in EST instead of animals in standard tests of toxicity. The embryotoxic potential of compounds (non, weak or strong embryotoxicity) may be obtained with a biostatistics-based prediction model and calculated from three different experimental endpoint values: The potency to inhibit growth of i) BALB/c-3T3 cells and ii) mES-D3 cells (IC503T3 and IC50ES) as presented using a cell cytotoxicity assay, and iii) the potency to inhibit differentiation of mES-D3 cells into contracting cardiomyocytes (ID50 D3) as demonstrated in a mES-D3 cell differentiation assay. In the present study, a model of EST with mES-D3 cells and BALB/c-3T3 cells was established, according to the standard EST system of the EU Center for the Validation of Alternative Methods, and verified it with 5-fluorouracil (strong embryotoxicity) as a positive control and penicillin G (non-embryotoxic) as a negative control. In addition, the authors further assessed the embryotoxicity of four compounds (eugenol, carnosic acid, procyanidin and dioctyl phthalate) with this model. The embryotoxic potentials of the four compounds were successfully classified by the EST system. Eugenol exhibited strong embryotoxicity, carnosic acid and dioctyl phthalate exhibited weak embryotoxicity, while procyanidin exhibited non-embryotoxicity.

Integrating in vitro data and physiologically based kinetic (PBK) modelling to assess the in vivo potential developmental toxicity of a series of phenols

Toxicity outcomes derived in vitro do not always reflect in vivo toxicity values, which was previously observed for a series of phenols tested in the embryonic stem cell test (EST). Translation of in vitro data to the in vivo situation is therefore an important, but still limiting step for the use of in vitro toxicity outcomes in the safety assessment of chemicals. The aim of the present study was to translate in vitro embryotoxicity data for a series of phenols to in vivo developmental toxic potency values for the rat by physiologically based kinetic (PBK) modelling-based reverse dosimetry. To this purpose, PBK models were developed for each of the phenols. The models were parameterised with in vitro-derived values defining metabolism and transport of the compounds across the intestinal and placental barrier and with in silico predictions and data from the literature. Using PBK-based reverse dosimetry, in vitro concentration–response curves from the EST were translated into in vivo dose–response curves from which points of departure (PoDs) were derived. The predicted PoDs differed less than 3.6-fold from PoDs derived from in vivo toxicity data for the phenols available in the literature. Moreover, the in vitro PBK-based reverse dosimetry approach could overcome the large disparity that was observed previously between the in vitro and the in vivo relative potency of the series of phenols. In conclusion, this study shows another proof-of-principle that the in vitro PBK approach is a promising strategy for non-animal-based safety assessment of chemicals.

In vitro developmental toxicity potencies of petroleum substances in the embryonic stem cell test as compared to their in vivo developmental toxicity potencies

In vitro developmental toxicity potencies of petroleum substances in the embryonic stem cell test as compared to their in vivo developmental toxicity potencies

Application of Mouse Embryonic Stem Cell Test to Detect Gender-Specific Effect of Chemicals: A Supplementary Tool for Embryotoxicity Prediction.

Gender effect is an inherent property of chemicals, characterized by variations caused by the chemical-biology interaction. It has widely existed, but the shortage of an appropriate model restricts the study on gender-specific effect. The embryonic stem cell test (EST) has been utilized as an alternative test for developmental toxicity. Despite its numerous improvements, mouse embryonic stem cells with an XX karyotype have not been used in the EST, which restricts the ability of the EST to identify gender-specific effects during high-throughput-screening (HTS) of chemicals to date. To address this, the embryonic stem cell (ESC) SP3 line with an XX karyotype was used to establish a "female" model as a complement to EST. Here, we proposed a "double-objects in unison" (DOU)-EST, which consisted of male ESC and female ESC; a seven-day EST protocol was utilized, and the gender-specific effect of chemicals was determined and discriminated; the replacement of myosin heavy chain (MHC) with myosin light chain (MLC) provided a suitable molecular biomarker in the DOU-EST. New linear discriminant functions were given in the purpose of distinguishing chemicals into three classes, namely, no gender-specific effect, male-susceptive, and female-susceptive. For 15 chemicals in the training set, the concordances of prediction result as no gender effect, male susceptive, and female susceptive were 86.67%, 86.67%, and 93.33%, respectively, the sensitivities were 66.67%, 83.33%, and 83.33%, respectively, and the specificities were 91.67%, 88.89%, and 100%, respectively; the total accuracy of DOU-EST was 86.67%. For three chemicals in the test set, one was incorrectively predicted. The possible reason for misclassification may due to the absence of hormone environment in vitro. Leave-one-out cross-validation (LOOCV) indicated a mean error rate of 18.34%. Taken together, these data suggested a good performance of the proposed DOU-EST. Emerging chemicals with undiscovered gender-specific effects are anticipated to be screened with the DOU-EST.

Prediction of in vivo developmental toxicity by combination of Hand1-Luc embryonic stem cell test and metabolic stability test with clarification of metabolically inapplicable candidates

Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST) is a promising alternative method for evaluation of developmental toxicity. However, the problems of predictivity have remained due to appropriateness of the solubility, metabolic system, and prediction model. Therefore, we assessed the usefulness of rat liver S9 metabolic stability test using LC–MS/MS to develop new prediction model. A total of 71 chemicals were analyzed by measuring cytotoxicity and differentiation toxicity, and highly reproducible (CV=20%) results were obtained. The first prediction model was developed by discriminant analysis performed on a full dataset using Hand1-Luc EST, and 66.2% of the chemicals were correctly classified by the cross-validated classification. A second model was developed with additional descriptors obtained from the metabolic stability test to calculate hepatic availability, and an accuracy of 83.3% was obtained with applicability domain of 50.7% (=36/71) after exclusion of 22 metabolically inapplicable candidates, which potentially have a metabolic activation property. A step-wise prediction scheme with combination of Hand1-Luc EST and metabolic stability test was therefore proposed. The current results provide a promising in vitro test method for accurately predicting in vivo developmental toxicity.

Comparison of embryotoxicity of di(2-ethylhexyl) phthalate using mouse and human embryonic stem cell test models in vitro

To establish a mouse embryonic stem cell test (mEST) model and human embryonic stem cell test (hEST) model, to evaluate the embryotoxicity of di(2-ethylhexyl) phthalate (DEHP). We developed mEST and hEST models according to the European Centre for the Validation of Alternative METHODS (ECVAM). We used penicillin G (PN-G) as the standard negative reference and 5-fluorouracil (5-FU) as the standard positive reference, respectively, to verify validity of the models. Based on model validity, mouse embryonic stem cells D3 (mESC-D3), mouse Balb/c-3T3 (3T3), and human embryonic stem cells H9 (hESC-H9) were administered different concentrations of DEHP (15.6, 31.2, 62.5, 125.0, 250.0, 500.0, and 1 000.0 μg/ml) for 7 days. A cell counting Kit-8 was used to detect the 50% inhibitory proliferation concentration (IC50) of mESC-D3 cells, 3T3 cells, and hESC-H9 with DEHP. mESC-D3 and hESC-H9 were treated with DEHP (15.6, 31.2, 62.5, 125.0, 250.0 μg/ml, and 500.0 μg/ml) for 10 days based on the cytotoxicity results. At day 10, the expression of cardiomyocyte differentiation gene alpha-myosin heavy chain (α-MHC) was detected by real-time PCR and the 50% inhibition of cardiomyocycte differentiation (ID50) determined. Based on the values of IC50 and ID50, functions Ⅰ, Ⅱ and Ⅱ could be calculated by three linear discriminant functions in the EST model and the embryotoxicity of DEHP described by comparing the three functions. Nontrophoblast lineage both ES cells were cultured under optimal conditions and highly expressed hESC markers OCT4 , SSEA4, and TRA-1-60. The embryoid bodies formed were uniform in size and shape, and these results were highly repeatable. The PN-G and 5-FU results coincided with the prediction by ECVAM. Validation of our EST models was satisfactory. RESULTS of the three endpoints of DEHP in mEST were 197.3 μg/ml (IC50 3T3), 210.0 μg/ml (IC50 D3) and 246.8 μg/ml (ID50 D3). DEHP was evaluated to be a nonembryotoxic compound based on values of function Ⅰ (7.78), function Ⅱ (7.58) and function Ⅲ (-7.79). The three endpoints of DEHP in hEST were 195.4 μg/ml (IC50 3T3), 184.8 µg/ml (IC50 D3), and 84.3 µg/ml (ID50). By comparing the values of function Ⅰ (3.21), function Ⅱ (5.77), and function Ⅲ (-6.46), DEHP was evaluated to be weakly embryotoxic. DEHP was determined to be a nonembryotoxic compound by mEST and weakly embryotoxic by hEST. Therefore, hEST is a more sensible model for the evaluation of DEHP embryotoxicity.

Development of technique for in vitro embryotoxicity of dental biomaterials.

SummaryThe Embryonic Stem Cell Test (EST) developed in Germany in 1997 is known as a screening test method capable of predicting the presence of unknown chemicals influencing normal human development. Firstly, we investigated the embryotoxicity of 24 types of monomer including dental monomers and dental alloy-component metal elements using this test. Monomers including Bis-GMA contained in base resin of composite resin exhibited weak embryotoxicity, and the toxicity level varied among dental alloy-component metal elements. It was clarified that metal ions eluted from currently sold dental alloys show no embryotoxicity. Then, we investigated a method that also considers human metabolic activity, which is not possible with the EST, in the results of embryotoxicity. In addition, an evaluation method using a hybrid culture system for hepatocytes and mouse ES cells and a method using oviduct or uterus cells for feeder cells were also investigated.

Editor's Highlight: Identification and Characterization of Teratogenic Chemicals Using Embryonic Stem Cells Isolated From a Wnt/β-Catenin-Reporter Transgenic Mouse Line.

Embryonic stem cells (ESCs) are commonly used for the analysis of gene function in embryonic development and provide valuable models for human diseases. In recent years, ESCs have also become an attractive tool for toxicological testing, in particular for the identification of teratogenic compounds. We have recently described a Bmp-reporter ESC line as a new tool to identify teratogenic compounds and to characterize the molecular mechanisms mediating embryonic toxicity. Here we describe the use of a Wnt/β-Catenin-reporter ESC line isolated from a previously described mouse line that carries the LacZ reporter gene under the control of a β-Catenin responsive promoter. The reporter ESC line stably differentiates into cardiomyocytes within 12 days. The reporter was endogenously induced between day 3-5 of differentiation reminiscent of its expression in vivo, in which strong LacZ activity is detected around gastrulation. Subsequently its expression becomes restricted to mesodermal cells and cells undergoing an epithelial to mesenchymal transition. The Wnt/β-Catenin-dependent expression of the reporter protein allowed quantification of dose- and time-dependent effects of teratogenic chemicals. In particular, valproic acid reduced reporter activity on day 7 whereas retinoic acid induced reporter activity on day 5 at concentrations comparable to the ones inhibiting the formation of functional cardiomyocytes, the classical read-out of the embryonic stem cell test (EST). In addition, we were also able to show distinct effects of teratogenic chemicals on the Wnt/β-Catenin-reporter compared with the previously described Bmp-reporter ESCs. Thus, different reporter cell lines provide complementary tools for the identification and analysis of potentially teratogenic compounds.

Growing knowledge of using embryonic stem cells as a novel tool in developmental risk assessment of environmental toxicants

Developmental toxicology is an important area of novel toxicology. In recent years, there have been big concerns toward the increasing exposure to pharmaceutical agents, food additives, pesticides, occupational toxicants, and environmental pollutants, as well as their possible association with all aspects of male or female-mediated transient or permanent defects in progeny. Therefore, it is of great importance to look for new predictive models to evaluate environmental toxicants before they can harm the human health and embryo development. In this regard, new cell-based in vitro screening models have been developed and validated in predictive toxicology to minimize assay costs and animal usage.Stem cell-based models have been increasingly applied for predicting the toxicity of chemicals. One of the most promising existing in vitro developmental toxicity tests is the validated embryonic stem cell test (EST) which employs marine or human embryonic stem cells to assess the potential of chemicals embryotoxicity. These cells are very suitable for embryotoxicity assessment as they have been demonstrated to specify cellular developmental processes during early embryogenesis and gene expression patterns of differentiation to functionally competent specialized cell types.The present paper aimed at criticizing the human and experimental evidence for developmental toxic effects of environmental toxicants based on ESCs models. Accordingly, pesticides, heavy metals, plasticizers, nanomaterials and some solvents have been considered as the main evaluated environmental toxicants inducing developmental toxicity. At the end, current challenges, pros and cons of using ESCs as an alternative validated in vitro model for specific developmental toxicity screening are discussed.

Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell.

The embryotoxic effect of intermediate frequency (IF) magnetic field (MF) was evaluated using murine embryonic stem (ES) cells and fibroblast cells based on the embryonic stem cell test (EST). The cells were exposed to 21 kHz IF–MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days) or the cell differentiation process (10 days) during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF–MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF–MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2, and an early developmental gene, Hba-x, in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH) cooktop, etc. in our daily lives, these results suggested that IF–MF in which the public is exposed to in general living environment would not have embryotoxic effect.

Establishment of a female mouse embryonic stem cell test to detect gender-specific effect: A supplementary tool for embryotoxicity prediction in vitro

Establishment of a female mouse embryonic stem cell test to detect gender-specific effect: A supplementary tool for embryotoxicity prediction in vitro

Comparative study of the zebrafish embryonic toxicity test and mouse embryonic stem cell test to screen developmental toxicity of human pharmaceutical drugs

Comparative study of the zebrafish embryonic toxicity test and mouse embryonic stem cell test to screen developmental toxicity of human pharmaceutical drugs

Impact of homeopathic remedies on the expression of lineage differentiation genes: an in vitro approach using embryonic stem cells

Well-documented studies of the potential effects and safety of homeopathic medicines in pregnancy are required. In this study, specific genes were studied which could serve as biomarkers for specification of three lineages to predict the safety of homeopathic remedies using mouse embryonic stem (ES) cells. Thus, the present work was to study the effects of homeopathic remedies taken during pregnancy using ES cells as the model. Mouse ES cells were exposed to 30C potency of Nux Vomica and Sepia, which are homeopathic medicines prescribed for the management of pregnancy related symptoms. Cytotoxicity studies were done using a modified Embryonic Stem cell test (EST). The expression levels of key genes and proteins were analyzed using real time polymerase chain reaction and immunocytochemistry, respectively. Homeopathic treatment led to modulations in the expression of certain lineage specific genes but this difference was not significant with respect to solvent control and showed normal differentiation as demonstrated by the expression of α/β MHC and α-actinin proteins in the differentiated ES cells. Our study for the first time has shown the feasibility of using ES cells in the developmental toxicity testing of remedies. The results suggest that they are not associated with developmental toxicity.

A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs on Early Embryogenesis.

Mammalian fetal development is easily disrupted by exogenous agents, making it essential to test new drug candidates for embryotoxicity and teratogenicity. To standardize the testing of drugs that might be used to treat pregnant women, the U.S. Food and Drug Administration (FDA) formulated special grade categories, labeled A, B, C, D and X, that define the level of risk associated with the use of a specific drug during pregnancy. Drugs in categories (Cat.) D and X are those with embryotoxic and/or teratogenic effects on humans and animals. However, which stages of pregnancy are affected by these agents and their molecular mechanisms are unknown. We describe here an embryonic stem cell test (EST) that classifies FDA pregnancy Cat.D and Cat.X drugs into 4 classes based on their differing effects on primitive streak formation. We show that ~84% of Cat.D and Cat.X drugs target this period of embryogenesis. Our results demonstrate that our modified EST can identify how a drug affects early embryogenesis, when it acts, and its molecular mechanism. Our test may thus be a useful addition to the drug safety testing armamentarium.

RNA transcripts for the quantification of differentiation allow marked improvements in the performance of embryonic stem cell test (EST).

Embryonic stem cell test (EST) is an in vitro validated assay for testing embryotoxicity. The EST needs improvements before being used for regulatory purposes, but also needs technical simplification for use in high throughput screenings. We propose the quantification in alterations of the differentiation of D3 monolayer cells cultures through the expression of biomarker genes in a shorter (5-day) and technically simpler (we use only monolayer cultures) test. We have defined a set of sixteen different genes biomarkers of ectoderm (Nrcam, Nes, Shh and Pnpla6), endoderm formation (Flk1 and Afp), mesoderm formation (Mesp1, Vegfa, Myo1e and Hdac7) and general cellular processes (Cdk1, Myc, Jun, Mixl, Cer and Wnt3). These, together with alterations in the viability of D3 and 3T3 cells and the prediction model of a classic EST, enhance the features of EST determinations to 100% concordance between in vivo-in vitro predictions with a set of seven different chemicals used in the validation of a classic EST. In conclusion, the proposed changes implemented in the classic EST confer it more reliability, speed and technical simplicity, which brings the EST closer to high throughput processes and regulatory purposes.