A <i>NANOG</i> - <i>EGFP</i> iPSC-Based Reporter Platform for High-Throughput Screening of Environmental Pollutant Effects on Pluripotency

Humans are frequently exposed to diverse environmental pollutants that may pose potential risks during early embryonic development. Advances in stem cell toxicology have introduced new opportunities for environmental toxicity assessment, enabling the development of physiologically relevant models based on human induced pluripotent stem cells (iPSCs). However, current systems remain limited by low throughput and the inability to achieve real-time monitoring. In this study, we established a NANOG-EGFP iPSC reporter line by precisely inserting a T2A-EGFP cassette into the endogenous NANOG locus using CRISPR/Cas9, enabling dynamic and quantitative visualization of pluripotency status. Using this reporter system, we systematically evaluated the effects of 38 representative environmental pollutants spanning six major chemical categories, including bisphenols, per- and polyfluoroalkyl substances (PFAS), halogenated flame retardants, organophosphate esters, neonicotinoids, and phenolic compounds. Most pollutants induced varying degrees of reduction in NANOG-EGFP expression, suggesting perturbation of core transcriptional networks involved in pluripotency maintenance rather than nonspecific cytotoxic effects. Notably, bisphenol B (BPB), bisphenol Z (BPZ), perfluorobutanesulfonic acid (PFBS), tributyl phosphate (TnBP), tris(2-chloroethyl) phosphate (TCEP), and 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHT-COOH) exhibited pronounced low-dose activity, causing significant suppression of NANOG-EGFP signals even at 10 nmol/L. Furthermore, during neuroectoderm differentiation, several bisphenols and PFAS delayed the normal downregulation of NANOG, indicating disruption of the tightly regulated timing of pluripotency exit and lineage transition during early developmental progression. Overall, the NANOG-EGFP iPSC reporter model provides a sensitive, stable, and practical platform for high-throughput screening of environmental toxicants and offers mechanistically informed insight into how pollutant exposure may interfere with early human developmental regulatory programs.