Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation

Abstract

The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicity in vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 μg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes: Pnpla6, Afp, Hdac7, Vegfa, and Nes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression of Nes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting for in vitro risk assessment embryotoxicity.

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

“…However, the HT capacity of current EST is hampered by its long test period, laborious protocol, and technical complexity of prediction model (Romero et al, 2011). However, the HT capacity of current EST is hampered by its long test period, laborious protocol, and technical complexity of prediction model (Romero et al, 2011).…”

“…This can significantly improve the accuracy of EST . Romero et al (2011) investigated several gene expressions derived from different embryo lineages, including Afp, Vegfa and Hdac7 as endoderm markers, Flk1 as mesoderm marker, and Nes as ectoderm marker, and used them to classify the embryotoxicity level of 5-fluorouracil, 5,5-diphenylhydantoin and saccharin. However, multiple endpoints using different gene markers for different differentiation lineages are complicate to use in HTS.…”

“…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). Using gene biomarkers of differentiation can further improve EST and shorten test duration to 5 days (Romero, Vilanova, & Sogorb, 2011). Using gene biomarkers of differentiation can further improve EST and shorten test duration to 5 days (Romero, Vilanova, & Sogorb, 2011).…”

“…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;. Using gene biomarkers of differentiation can further improve EST and shorten test duration to 5 days (Romero, Vilanova, & Sogorb, 2011). Several modified ESTs analyzing gene biomarkers specific to different differentiation lineages including cardiomyocytes (Nagahori, Suzuki, Coz, Omori, & Saito, 2016;Skelton, Kamp, Elliott, & Ardehali, 2017;Suzuki, Ando, Yamashita, et al, 2011), neural cells (Kobayashi et al, 2017;Yu et al, 2015), and osteoblasts (Sittner, Huhse, Steinfath, Luch, & Seiler, 2016) as molecular endpoints have been developed.…”

A Developmental Toxicology Assay Platform for Screening Teratogenic Liability of Pharmaceutical Compounds

“…The EST and WEC assays were further explored with new assays developed higher in throughput with automation potential. Such efforts for increasing throughput include molecular-based embryonic stem cell assays that use flow cytometry, reporter constructs, or transcriptional endpoints in their screening protocols (Bigot et al, 1999;zur Nieden et al, 2001zur Nieden et al, , 2004Seiler et al, 2002Seiler et al, , 2004Buesen et al, 2009;van Dartel et al, 2009van Dartel et al, , 2010avan Dartel et al, , 2010bvan Dartel et al, , 2011Barrier et al, 2011;Romero et al, 2011;Suzuki et al, 2011;Panzica-Kelly et al, 2013). In addition, development of a streamlined rat WEC assay provided a morphological assay with reduced animal usage and improved throughput and results turnaround .…”