Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST): A novel rapid and highly reproducible <i>in vitro</i> test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells

Abstract

-The embryonic stem cell test (EST) is a promising alternative method for evaluating embryotoxicity of test chemicals by measuring cytotoxicity and differentiation toxicity using mouse ES cells. Differentiation toxicity is analyzed by microscopically counting the beating of embryonic bodies after 10 days of culture. However, improvements are necessary to reduce the laborious manipulations involved and the time required to obtain results. We have previously reported the successful stable transfection of ES cells (ES-D3) with the heart and neural crest derivatives expressed transcript 1 (Hand1) gene and the establishment of a 96-well multi-plate-based new EST with luciferase reporter assay 6 days after treatment with test chemicals. Now, we propose an even more rapid and easier EST, named Hand1-Luc EST. We established another cell line to monitor the Hand1 gene expression via a luciferase reporter gene. By mRNA analysis and luciferase assay, we examined in detail the luciferase activity during cell differentiation, which allowed us to reduce the time of measurement from day 6 to day 5 (120 hr). Furthermore, the protocol was improved, with, among others, the measurement of cytotoxicity and differentiation toxicity taking place in the same 96-well round bottom plate instead of two different plates. With the positive control, 5-fluorouracil (5-FU), and 9 test chemicals, data with high reproducibility and very low variation (CV < 50%) in the relevant endpoints were obtained. This study shows that the Hand1-Luc EST could provide an accurate and sensitive short-term test for prediction of embryotoxicants by measuring cytotoxicity and differentiation toxicity from the same sample.

An engineered mouse embryonic stem cell model with survivin as a molecular marker and EGFP as the reporter for high throughput screening of embryotoxic chemicals in vitro

“…boric acid at 100 µg/ml and methoxyacetic acid (MAA) at 180 µg/ml, EGFP fluorescence intensities from D3-CMV-EGFP and D3-SP-EGFP cells were lower compared to the control, indicating a cytotoxic effect. Meanwhile, the specific survivin expression decreased~20% in 3 days (Figure 5), indicating a moderate embryotoxic effect, which was consistent with the EST result for boric acid and MAA as weakly embryotoxic compoundsSuzuki, Ando, Yamashita, et al, 2011;Le Coz, Suzuki, Nagahori, Omori, & Saito, 2015).…”

“…(b) Fluorescence images for 6 and 30 days old EBs formed by D3-CMV-EGFP cells. Meanwhile, the specific survivin expression decreased~20% in 3 days(Figure 5), indicating a moderate embryotoxic effect, which was consistent with the EST result for boric acid and MAA as weakly embryotoxic compoundsSuzuki, Ando, Yamashita, et al, 2011;Le Coz, Suzuki, Nagahori, Omori, & Saito, 2015). EBs: embryoid bodies; EGFP: enhanced green fluorescent protein [Color figure can be viewed at wileyonlinelibrary.com] saccharin and Penicillin G are noncytotoxic and nonembryotoxic withreported IC 50 and ID 50 values greater than 1000 μg/mlSuzuki, Ando, Yamashita, et al, 2011;Scholz et al, 1999).…”

Integration of read-across and artificial neural network-based QSAR models for predicting systemic toxicity: A case study for valproic acid

“…Susukida et al, 2016) or developmental toxicity (e.g. Le Coz et al, 2015), are available, and should serve to increase the accuracy of hazard identification.…”

Prospects and Frontiers of Stem Cell Toxicology

“…In another example, groups of genes representative of the whole genome and that were involved in toxicity responses were selected for more predictive, reliable, and yet less laborious assays [ 10 , 20 , 29 ]. More recently, a shortened EST based on the detection of the cardiac and neural crest Hand1 gene expression, through a luciferase reporter assay, showed good reproducibility [ 22 ]. In addition, studies on the effects of flusilazole and monophthalates on mESC cardiogenesis convinced that low chemical concentrations, although not cytotoxic, compromised mESC cardiogenesis by downregulating the expression of related genes in a dose-dependent manner [ 20 , 30 ].…”