Innovative approaches in the embryonic stem cell test (EST)

Abstract

The embryonic stem cell test (EST) is a high-throughput in vitro screening assay for developmental toxicity free of animal use. The EST uses the ability of murine embryonic stem cells to differentiate into the mesodermal cardiac lineage in combination with two cytotoxicity test systems. Validation of the EST showed that the test system is very promising as an alternative method to animal testing, however to optimize predictability and increase knowledge on the applicability domain of the EST, improvements to the method were proposed and studied. In this review we discuss the first definition of the EST followed by the innovative approaches which have been proposed to increase the predictivity of the EST, including implementation of molecular endpoints in the EST, such as omics technologies and the addition of alternative differentiation models to the testing paradigm, such as neural and osteoblast differentiation and the use of human stem cells. These efforts to improve the EST increase the value of embryonic stem cells used as in vitro systems to predict developmental toxicity.

The 3R principle: advancing clinical application of human pluripotent stem cells

“…In doing so, this approach goes a significant way towards defining the suitability of human PS cell-derived RPE for both research and clinical applications. Future development of similar assay panels for other differentiated cell types will enable a reduction in the use of animals in research and, equally importantly, will enable robust phenotypic and functional cell characterisation that increases confidence in the data generated [9]. …”

“…However, significant international interest from scientists, ethicists, and regulatory bodies is driving the development of replacement in vitro assays [37,39]. For example, the mouse EST has already been validated by the European Centre for the Validation of Alternative Methods as a viable alternative assay for embryotoxicity [9,10]. This 7 to 10-day assay makes use of undifferentiated ES cells, ES cell-derived cardiomyocytes, and NIH 3T3 cells, together with statistical assessment of endpoints that include cell morphology, viability, beating frequency, and more recently gene expression and proteomic analyses.…”

“…As described below, the translation of PS cell technology from academic research to biotechnology applications has already begun. This includes use of the embryonic stem cell test (EST) to predict embryotoxicity [9] and clinical trials using human ES cell-derived oligodendrocytes and retinal pigment epithelium (RPE) [11,12]. These early successes suggest that further application of the 3R principle to human PS cell research will be economically, clinically, and ethically useful.…”

Developmental toxicity assessment of common excipients using a stem cell-based in vitro morphogenesis model

“…Because of their embryo-like properties, pluripotent stem cells have been explored as non-animal alternatives to study developmental toxicity. In assays commonly referred to as embryonic stem cell tests (ESTs), the potential developmental toxicity of compounds is evaluated based on their inhibitory effects on the in vitro differentiation of ES cells as well as their cytotoxic impact on ES cell and somatic cell lines, specifically NIH/3T3 fibroblast (Riebeling et al, 2012; Theunissen and Piersma, 2012). The original EST, or ESTc, measures concentrations of a test compound that inhibit cardiomyocyte differentiation by 50% (ID50) as well as concentrations that inhibit proliferation of ES and NIH/3T3 cells by 50% (D3 IC50 and 3T3 IC50, respectively).…”

Polymer Biocompatibility

“…The authors discuss the first definition of the embryonic stem cell test and the innovative approaches that have been proposed to increase the predictivity of the embryonic stem cell test, including implementation of molecular end points in the embryonic stem cell test, such as OMICS technologies and the addition of alternative differentiation models to the testing paradigm, such as neural and osteoblast differentiation and the use of human stem cells. These efforts to improve the embryonic stem cell test increase the value of embryonic stem cells used as in vitro systems to predict developmental toxicity (Theunissen & Piersma, 2012).…”