Elke Genschow

Embryotoxicity Screening Using Embryonic Stem Cells in vitro: Correlation to in vivo Teratogenicity

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. The embryonic stem cell test that makes use of the differentiation of ES cells into cardiomyocytes in a standardized in vitro model was developed to offer an alternative method to comprehensive in vivo studies in reproductive toxicology about toxic effects of chemicals. ES cells of the mouse cell line D3 are investigated for their preserved capability to differentiate following drug exposure, and both ES cells and differentiated fibroblast cells of the mouse cell line 3T3 are comparatively analyzed for effects on viability. The following endpoints are used to classify the embryotoxic potential of chemicals into three classes of in vitro embryotoxicity (non-, weakly or strongly embryotoxic). These endpoints are: (1) the inhibition of differentiation of ES cells into cardiomyocytes after 10 days of treatment, and the decrease of viability (cytotoxicity) of (2) 3T3 cells and (3) ES cells after 10 days of treatment, determined by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) test. 50% inhibition concentrations for differentiation (ID50) and cytotoxicity (IC50D3 and IC503T3) are calculated from concentration-response curves. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed. This procedure identified three variables, the lg(IC50D3), the lg(IC503T3) and the relative distance between IC503T3 and ID50, that improved the separation of the three classes of embryotoxicity compared to the prediction model that was originally proposed after test development. Unlike the orginal PM, the improved PM incorporates as one variable the relative distance between IC503T3 and ID50, instead of the ratio ID50/IC50D3 that was used previously.

Prevalidation of the Embryonic Stem Cell Test (EST)-A New In Vitro Embryotoxicity Test.

Pluripotent embryonic stem cells (ES cells) of the mouse (cell-line D3) can be maintained in the undifferentiated state in the presence of LIF (Leukaemia Inhibitory Factor). Upon withdrawal of LIF, these cells differentiate into various cell types under appropriate conditions. This property of ES cells allowed us to develop an in vitro embryotoxicity test, the Embryonic Stem Cell Test (EST; In Vitro Toxicology 1997, 10, 119-127), which does not require taking embryonic cells or tissues from pregnant animals. In the EST, the effect of test chemicals on three endpoints is assessed: inhibition of the differentiation of ES cells into contracting myocard, cytotoxicity in ES cells and cytotoxicity in mouse 3T3 fibroblasts, which are serving as differentiated cells in the test. The results of a prevalidation study of the EST are described, which was conducted according to the ECVAM prevalidation scheme. In the first stage of the study (Phase I), a standard operating procedure (SOP) was elaborated. In the second phase (Phase II), the interlaboratory transferability of the EST was assessed using three test chemicals representing three classes of embryotoxicity (a strong, a weak and a non-embryotoxic chemical) in two European laboratories (ZEBET at the BgVV in Berlin, Germany; ECVAM at the JRC in Ispra, Italy) and one US laboratory (Institute for In Vitro Sciences (IIVS) in Gaithersburgh, MA, USA). In the final stage of prevalidation (Phase III), nine test chemicals and a positive control were tested under blind conditions at ZEBET and ECVAM. The statistical evaluation of the results led to the development of an improved prediction model for 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.

Permanent embryonic germ cell lines of BALB/cJ mice--an in vitro alternative for in vivo germ cell mutagenicity tests.

To offer a sensitive and predictive in vitro method to assess germ cell mutagenicity, we established primordial germ (PG) cell-derived permanent female and male embryonic germ (EG) cell lines of the mouse (strain BALB/cJ). The differences in developmental sensitivity of EG cells and differentiated fibroblast cells of the mouse cell line 3T3 to genotoxicants were tested comparatively under identical test conditions. Cytotoxicity assay was measured by the MTT test and genotoxic effects were determined by sister chromatid exchanges (SCE) rates induced by standard reference mutagens. Both methods are used to assign the chemicals to two classes of in vivo reproductive toxicity, non- and strongly genotoxic to germ cells. Applying linear discriminant analysis, a biostatistical prediction model (PM) was developed for the female cell line EG(3). This procedure identified a single variable, the Ig(SCE(200)EG(3)) as the statistically significant concentration related increase of 200% in the mean number of SCEs per metaphase spread after 3 h of exposure to be sufficient for separation into the classes: non- and strongly genotoxic to germ cells. Applying this PM to the training set of five genotoxic and three non-genotoxic test chemicals, 100% correct classifications were obtained.