Improvement of the embryonic stem cell test endpoint analysis by use of field potential detection

Abstract

-The embryonic stem cell test (EST) is a validated in vitro method to assess the embryotoxic potential of compounds and is a promising tool for drug screening. EST requires microscopic observation of beating cardiomyocytes differentiated from embryonic stem cells as a toxicological endpoint. However, this process is time-consuming and lacks throughput performance. To improve the analysis, we introduced an electrophysiological method with a microelectrode array system for the evaluation of differentiated cardiomyocytes. Embryotoxic (valproic acid, verapamil, and 5-fluorouracil) and non-embryotoxic (penicillin G, d-camphor, and isoniazid) compounds were assessed with the system. Mouse embryonic stem cells were differentiated into cardiomyocytes and treated with each compound during the differentiation process. The embryotoxicity of each compound was then assessed by measuring the field potentials of differentiated cardiomyocytes using the microelectrode array system, as well as by microscopic evaluation. All the embryotoxic compounds dose-dependently inhibited the field potential formation and the myocardial beating of differentiated cells, while the non-embryotoxic compounds did not affect either endpoint. The detection capabilities of the two assay methods were similar. These results indicated that the field potential measurements can be used as an alternative endpoint of EST. Moreover, the field potential can be measured automatically, introducing a high throughput performance compared to the conventional microscopic observation. We therefore concluded that the endpoint analysis with the microelectrode array system improves the original EST and can be useful for the assessment of the embryotoxic potential of compounds.Key words: Cardiomyocyte, Embryonic stem cell, Embryonic stem cell test (EST), Embryotoxicity, Field potential, Microelectrode array system Correspondence: Naoteru Koseki (E-mail: naoteru-koseki@ds-pharma.co.jp) Original ArticleThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.35, No.5, 619-629, 2010 Vol. 35 No. 5 619Recently, examinations using embryonic stem cells (ES cells) have been reported as novel in vitro assay methods for the screening of embryotoxicity (Scholz et al., 1999;Pellizzer et al., 2005). The embryonic stem cell test (EST) has been validated by the European Centre for the Validation of Alternative Methods (ECVAM) to assess the embryotoxic potential of compounds and its results are reported to correlate well with those of in vivo methods . In EST, the ES cells are exposed to a test compound throughout the entire differentiation stage and therefore the effects of the compound can be assessed throughout the period of organogenesis. Thus, it is thought that EST is suitable for in vitro early drug screening for embryotoxicity.On the other hand, some serious problems have been noted for the original EST, especially in the area of sufficient throughput performance. The most serious problem of EST is mainly caused by its endpoint analysis. EST requires the microsc...

Usefulness of field potential as a marker of embryonic stem cell-derived cardiomyocytes, and endpoint analysis of embryonic stem cell test

“…The changes of these parameters in 5-FU treated EBs were almost same as in previous papers Seiler et al, 2004;Koseki et al, 2010). Furthermore, the study also confirmed the relationship between expression area of cTnT and field potential signal or beating in these in 5-FU treatment groups, and concordance rate of these parameters was more than 70%.…”

“…EST, however, requires microscopic observation of beating cells to be considered cardiomyocytes differentiated from ES cells as an endpoint assay; this process is time-con- suming and a restricting factor for throughput performance, and some alternative methods to this endpoint assay have been developed (Bremer et al, 2001;Buesen et al, 2009;Peters et al, 2008;Seiler et al, 2004). In a previous study, we established a novel method to evaluate the differentiation of ES cells based on a field potential formulation for cardiomyocytes (Koseki et al, 2010). This field potential method can be performed automatically and can improve the throughput performance compared to the original EST method.…”

“…EST requires the microscopic observation of beating cells to be considered cardiomyocytes differentiated from embryonic stem (ES) cells as an endpoint assay, and this microscopic observation has been a limiting factor of this evaluation method and an obstacle to high-throughput screening. To resolve this problem, we established a method for the evaluation of differentiated cardiomyocytes based on the field potential, instead of the microscopic observation of beating (Koseki et al, 2010). As field potential signal was well correlated with that of microscopic endpoints and this field potential method can be conducted under high throughput, we consider that this "modified" EST method using field potential as an endpoint assay might be more practical at an early stage of drug screening.…”

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.…”

Application of zebrafish to safety evaluation in drug discovery

“…In addition, alternatives to animal testing have been actively sought from the viewpoint of the 3Rs principle for a more ethical use of animals in testing. To date, whole embryo culture (WEC) 17 and embryonic stem cell test (EST) 18 have been established as in vitro assay systems with modest throughput. However, these assay systems are not commonly used in drug discovery, because the culture period (of up to 3 days) prevents the use of WEC for evaluating the entire organogenesis process and EST has only been used to evaluate differentiation into cardiomyocytes.…”