The Validated Embryonic Stem Cell Test with Murine Embryonic Stem Cells

Abstract

Birth defects are the leading cause of infant mortality in the USA, yet the causes of most of these conditions are unknown. While a combination of genetic and environmental factors are suspected in most cases, little information exists about the health risks that prenatal exposure to many common chemicals poses for the fetus. Thus, development and refinement of procedures that can accurately predict embryotoxicity of compounds is important for curtailing the number of infants born with birth defects. The embryonic stem cell test (EST) is a procedure that utilizes comparison of cytotoxicity in embryonic and adult cells and inhibition of differentiation to predict embryotoxicity of compounds tested. Because of its use of existing cell lines, the EST dramatically reduces the need for animal test subjects in toxicity testing. In addition, because of its use of inhibition of differentiation as an endpoint, the EST is extremely versatile in the range of complications it can test for. In this chapter, procedures for use of the validated embryonic stem cell test with the traditional cardiomyocyte differentiation endpoint are explained. The protocol includes discussion of routine stem cell culture, the cardiomyocyte differentiation procedure, and methods for utilization of molecular endpoints for assessing embryotoxicity of compounds.

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

“…The EST validation study using 20 compounds with known in vivo embryotoxicity showed an overall accuracy of 78%. Although EST has been validated as a relatively reliable and reproducible tool to evaluate developmental toxicity or embryotoxicity (Genschow, Scholz, & Brown, 2000;Walker, McClelland-Descalzo, & zur Nieden, 2018), its application is limited by assay complexity, long assay duration (10 days), and morphological assessment of contracting cardiomyocytes Luz & Tokar, 2018), which is difficult to quantify and unable to assess embryotoxic effects on embryonic stem cell (ESC) differentiation to other lineages (Jong, Beek, & Piersma, 2014). FACS-EST using highly predictive protein markers specific for developing heart tissue to replace the morphological assessment can provide a better quantitative endpoint and shorten the assay time to 7 days (Seiler & Spielmann, 2011;.…”