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

Abstract

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.

Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing

“…Our data on thalidomide support this notion and indicated that our luminescence-based assay may have a high sensitivity and improved assay performance. Other reporter cell lines for developmental toxicity testing based on a fluorescence readout or β-galactosidase staining did not show increased sensitivity compared to the conventional readout of beating cardiomyocytes (Kugler et al 2015 , 2016 ). Furthermore, luminescence assays are less susceptible to autofluorescent chemicals than fluorescence-based assays (Fan and Wood 2007 ).…”

Pluripotent stem cell assays: Modalities and applications for predictive developmental toxicity

“…Another strategy used to generate transgenic ES cell lines is to isolate ESCs from transgenic mouse models, which has led to the generation of two reporter mESC lines that can be used to monitor Wnt and TGF-βb signaling ( Kugler et al, 2017 , Kugler et al, 2016 ). Both models have been tested with a small set of developmental toxicants (valproic acid, retinoic acid, and 6-aminonicotinamide), and reporter activity correlates well with subsequent cardiomyocyte differentiation assays, demonstrating the validity of these approaches for the small set of chemicals tested.…”

Pluripotent Stem Cells in Developmental Toxicity Testing: A Review of Methodological Advances

“…Another strategy used to generate transgenic ESC lines is to isolate ESCs from transgenic mouse models, which has led to the generation of 2 transgenic mESC lines that can be used to monitor Wnt and TGF-b signaling (Kugler et al, 2015(Kugler et al, , 2016. Both models have been tested with a small set of developmental toxicants (valproic acid, retinoic acid, and 6-aminonicotinamide), and reporter activity correlates well with subsequent cardiomyocyte differentiation assays, demonstrating the validity of these approaches.…”