Evaluation of Novel High-Throughput Embryonic Stem Cell Tests with New Molecular Markers for Screening Embryotoxic Chemicals In Vitro

Abstract

The embryonic stem cell test (EST) is a validated in vitro alternative test for prediction of embryotoxicity with inhibition of cardiomyocyte differentiation under the microscope as beating areas at day 10 as an endpoint. However, improvements are necessary for regulatory acceptance and application to high-throughput screening. We have previously reported that heart and neural crest derivatives expressed transcript 1 (Hand1), a transcription factor essential for mammalian heart development, and cardiomyopathy associated 1 (Cmya1), an intercalated disk protein implicated in cardiac morphogenesis, are quantitative and objective molecular endpoints for predicting embryotoxicity, detected at day 6 when mouse embryonic stem (ES) cells differentiate into cardiomyocytes. In established stable transgenic ES cells with Hand1 or Cmya1 promoters upstream of luciferase reporter gene, changes in each gene expression were found to be coincident with those in luciferase activities during cardiomyocyte differentiation, suggesting that monitoring might be possible by chemiluminescent determination. In our novel EST, differentiation toxicity and cytotoxicity of test chemicals were here analyzed using ES cells and 3T3 fibroblasts by this approach in 96-microwell plates. Extensive investigations were performed to explore predictive power and validity by comparing a set of 24 well-known test chemicals. The novel EST offers high predictability and accuracy with a reduced test duration and manpower compared with the original EST protocol, thus providing a new rapid and sensitive in vitro method for screening embryotoxicants.

Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (Hand1- and Cmya1-ESTs): a preliminary cross-laboratory performance analysis

“…The test chemicals were here classified by the PM reported previously (Genschow et al, 2004) using three endpoints (Table 2). In our previous study (Suzuki et al, 2011b), HU, a strong embryotoxic compound, was misclassifed as ''weak'' with both ESTs. In the present study, HU was also classified as "weak" with both ESTs in three laboratories.…”

“…Differentiation toxicity was analyzed by detecting chemical-related changes in Hand1 and Cmya1 promoter derived luminescence during the differentiation of Hand1-and Cmya1-ES cells into cardiomyocytes, as described in a previous report (Suzuki et al, 2011b). Briefly, cells in 50 μl of differentiation medium (750 cells/well) were seeded in white round bottom low attachment 96-well plates (Sumitomo Bakelite, Co. Ltd., Tokyo, Japan).…”

“…For screening purposes, the current experimental procedure of the EST with visual assessment of contracting cardiomyocytes requires extensive experimental expertise and is time-consuming with a rather long test duration of 10 days. Previously, we identified several marker genes as specifically altered by embryotoxic chemicals (Suzuki et al, 2011a) and also reported the establishment of 96 well multi-plate based new ESTs with Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (Hand1-and Cmya1-ESTs): a preliminary cross-laboratory performance analysis luciferase reporter assays using stable transgenic ESCs for detecting molecular markers (Hand1: heart and neural crest derivatives expressed transcript 1 or Cmya1: cardiomyopathy associated 1) (Suzuki et al, 2011b). The newly developed Hand1-and Cmya1-ESTs provide new rapid and sensitive in vitro methods for screening embryotoxicants.…”

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

“…In this context and in response to all of the current problems, our laboratory developed a novel high-throughput EST in 2011 (Suzuki et al, 2011b). In that study, we used the same type of cells as used in the original EST (D3 and 3T3 cells).…”

Improvement of the evaluation method for teratogenicity using zebrafish embryos

“…These study systems, however, are not appropriate for early-stage drug screening because they are low-throughput and time-consuming. Therefore, some alternative in vitro methods, e.g., embryonic stem cell test (EST: (Scholz et al, 1999)), mammalian micromass (MM) test (Flint, 1993), and whole embryo culture (WEC) test (Webster et al, 1997) have been validated by the European Centre for the Validation of Alternamethods have been reported (Peters et al, 2008;Koseki et al, 2010;Suzuki et al, 2011) to achieve more efficient throughput ability. Although these in vitro methods are useful for prediction of embryotoxicity or teratogenicity, drug exposure timing of MM or WEC test does not cover the whole period of organogenesis, and EST methods do not consider the complexity of embryotoxic mechanisms other than cardiac development.…”