US EPA

Environmental chemical exposures are a contributing factor to birth defects affecting infant morbidity and mortality. The USA EPA is committed to developing new approach methods (NAMs) to detect chemical risks to susceptible populations, including pregnant women. NAM-based coverage for cellular mechanisms associated with early human development could enhance identification of potential developmental toxicants (DevTox) for new and existing data-poor chemicals. The human pluripotent stem cell test (hPST) is an in vitro test method for rapidly identifying potential human developmental toxicants that employs directed differentiation of embryonic stem cells to measure reductions in SOX17 biomarker expression and nuclear localization. The objective of this study was to expand on the hPST principles to develop a model platform (DevTox GLR) that utilizes the transgenic RUES2-GLR cell line expressing fluorescent reporter fusion protein biomarkers for SOX17 (endoderm marker), BRA (mesoderm marker), and SOX2 (ectoderm and pluripotency marker). Initial assay adaption to definitive endoderm (DevTox GLR-Endo) was performed to emulate the hPST SOX17 endpoint and enable comparative evaluation of concordant chemical effects. Assay duration was reduced to two days and screening throughput scaled to 384-well format for enhanced speed and efficiency. Assay performance for 66 chemicals derived from reference and training set data resulted in a balanced accuracy of 72% (79% sensitivity and 65% specificity). The DevTox GLR-Endo assay demonstrates successful adaptation of the hPST concept with increased throughput, shorter assay duration, and minimal endpoint processing. The DevTox GLR model platform expands the in vitro NAM toolbox to rapidly identify potential developmental hazards and mechanistically characterize toxicant effects on pathways and processes associated with early human development.

Birth defects impact approximately 3% of births in the United States annually and are a major contributor to infant morbidity and mortality. The majority of developmental anomalies are of unknown etiology but there is increasing evidence that exposure to certain environmental chemicals is a contributing factor. Identifying prenatal developmental toxicants is challenging since target effects can span critical stages of fetal development (e.g. conception through organogenesis), resulting in adverse outcomes including low birth weight, congenital defects, functional deficits, and pregnancy loss. Key provisions in the Frank R. Lautenberg Chemical Safety for the 21st Century Act promote the use of non-animal, new approach methods (NAMs) to identify chemical risks to susceptible populations including infants, children, and pregnant women. Development and application of human in vitro NAMs that recapitulate defined endpoints of early embryonic development are necessary to address these challenges.  The human Pluripotent Stem Cell Test (hPST) utilizes directed endoderm differentiation of human stem cells to identify developmental toxicants that perturb SRY-box transcription factor 17 (SOX17) expression and nuclear localization. The objective of this study was to adapt the principles of the hPST assay for increased throughput, decreased assay duration, and broader mechanistic evaluation. The RUES2-GLR (germ layer reporter) human pluripotent stem cell line expands the biomarker profile to all three gastrulation lineages, thereby establishing the multi-lineage DevTox GLR model platform. The RUES2-GLR cell line has been transgenically modified by CRISPR-Cas9 technology to endogenously express fluorescent reporter fusion protein biomarkers from native gene loci for SOX17 (endoderm marker), Brachyury [BRA] (mesoderm marker), and SRY-Box Transcription Factor 2 [SOX2] (ectoderm and pluripotency marker). The cell line enables SOX17, BRA, and SOX2 protein expression evaluation without the need for fluorescent antibodies and provides additional mechanistic insight into pluripotency and differentiation pathways.   EPA and international OECD test guidelines for developmental and reproductive toxicity mandate the use of animal models to characterize both the hazard and risk of potential toxicants. In vivo animal testing is a time- and labor-intensive process that is costly, has limited testing throughput, and maintains difficulty in extrapolation of animal to human developmental effects. Given the high-throughput capacity, low cost, and reasonable predictive accuracy (72%), the DevTox GLR-Endo assay is a promising approach toward rapidly identifying and ascribing mechanisms of chemical toxicity associated with human development while being mindful of animal welfare.  

Gamble, J., K. Hopperstad, AND C. Deisenroth. The DevTox Germ Layer Reporter Platform: An Assay Adaptation of the Human Pluripotent Stem Cell Test. Toxics. MDPI, Basel, SWITZERLAND, 10(7):392, (2022). [DOI:10.3390/toxics10070392]