Trends in improving the embryonic stem cell test (EST): an overview

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 ES

Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests

An ECVAM-funded prevalidation study (PV) was conducted during 1999 and 2000 to identify in vitro tests capable of reliably distinguishing between skin irritants (I) and non-irritants (NI) according to European Union risk phrases ("R38" or no classification). The tests evaluated were EpiDerm, EPISKIN, PREDISKIN, the non-perfused pig ear method, and the mouse skin integrity function test (SIFT). Whereas reproducibility of the two human skin model tests and SIFT was acceptable, none of the methods was deemed ready to enter a formal validation study due to their low predictivity. The ECVAM Skin Irritation Task Force therefore suggested improvements of protocols and prediction models for these tests. Furthermore, it was agreed that experience gained with the two human-skin models be shared, and a common protocol should be developed for EpiDerm and EPISKIN (Zuang et al., 2002). When we applied an improved EPISKIN protocol (Portes et al., 2002) to the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was far too low. In 2003, the EPISKIN protocol was further refined by extension of the post-incubation period following chemical exposure. In the current study, we evaluated this EPISKIN refinement by applying it to EpiDerm. In addition, we developed technical improvements for the application of the test chemicals and rinsing procedure, which reduced the variability of results and increased the percentage of correct predictions. A set of twenty non-coded reference substances from the ECVAM prevalidation study phase III (Fentem et al., 2001) was tested with the final protocol in three independent runs. Both high sensitivity (80%) and high specificity (78%) were achieved, and the statistical probability of correct classifications was high, so that the test is now regarded ready for formal validatio

Evaluation of 309 Environmental Chemicals Using a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity Assay

The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC50) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation

Colour as a cue to eat : effects of plate colour on snack intake in pre-school children

Environmental cues, such as the colour of food and dishware, have been shown to influence food and drink consumption in adult populations. This proof of concept study investigated whether plate colour could be utilised as a strategy to reduce intake of high energy density (HED) snacks and increase intake of low energy density (LED) snacks in pre-school children. In a between and within-subjects design, children were randomly assigned to either a control group (no colour message) or intervention group (received a colour message: red = stop, green = go) and were provided a snack at nursery on three occasions on differently coloured plates (red, green and white), for each snack type (HED, LED). Snack intake, colour preference, colour association, and anthropometrics were recorded for each child. The results showed that there was no effect of group (control vs intervention) on HED (p=0.540) and LED intake (p=0.575). No effect of plate colour on HED (p=0.147) or LED snack intake (p=0.505) was evident. Combining red and green plates for a chromatic versus achromatic comparison showed that there was no significant effect of chromatic plate on HED (p=0.0503) and LED (p=0.347) intakes. Despite receiving a brief learning intervention, the use of plate colour was found in the present study to be an ineffective strategy to control snack food intake in pre-school aged children. Rather, we suggest that food intake in young children may best be predicted by portion size, energy density and eating behaviour traits

Workgroup Report: Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity into International Hazard and Risk Assessment Strategies

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19–21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work

Relativity in social cognition: basic processes and novel applications of social comparisons

A key challenge for social psychology is to identify unifying principles that account for the complex dynamics of social behaviour. We propose psychological relativity and its core mechanism of comparison as one such unifying principle. Social cognition is relative in that it is shaped by comparative thinking. If comparative thinking is indeed a central mechanism in social psychology, then it should be affected by, and affect itself, a wide variety of phenomena. To support our proposal, we review recent evidence investigating basic processes underlying and novel applications of social comparisons. Specifically, we clarify determinants of assimilation and contrast, evaluative consequences of comparing similarities vs. differences, attitudinal effects of spatial relativity, and how spatial arrangements determine perceived similarity, one of the antecedents of social comparisons. We then move to behavioural relativity effects on motivation and self-regulation, as well as imitation behaviour. Finally, we address relativity within the more applied areas of morality and political psychology. The reviewed research thereby illustrates how unifying principles of social cognition may be instrumental in answering old questions and discovering new phenomena and explanations

The Replication Database:Documenting the Replicability of Psychological Science

In psychological science, replicability—repeating a study with a new sample achieving consistent results (Parsons et al., 2022)—is critical for affirming the validity of scientific findings. Despite its importance, replication efforts are few and far between in psychological science with many attempts failing to corroborate past findings. This scarcity, compounded by the difficulty in accessing replication data, jeopardizes the efficient allocation of research resources and impedes scientific advancement. Addressing this crucial gap, we present the Replication Database (https://forrt-replications.shinyapps.io/fred_explorer), a novel platform hosting 1,239 original findings paired with replication findings. The infrastructure of this database allows researchers to submit, access, and engage with replication findings. The database makes replications visible, easily findable via a graphical user interface, and tracks replication rates across various factors, such as publication year or journal. This will facilitate future efforts to evaluate the robustness of psychological research