Neuronal and cardiac toxicity of pharmacological compounds identified through transcriptomic analysis of human pluripotent stem cell-derived embryoid bodies

Abstract

Concurrent with the ‘3R’ principle, the embryonic stem cell test (EST) using mouse embryonic stem cells, developed in 2000, remains the solely accepted in vitro method for embryotoxicity testing. However, the scope and implementation of EST for embryotoxicity screening, compliant with regulatory requirements, are limited. This is due to its technical complexity, long testing period, labor-intensive methodology, and limited endpoint data, leading to misclassification of embryotoxic potential. In this study, we used human induced pluripotent stem cell (hiPSC)-derived embryoid bodies (EB) as an in vitro model to investigate the embryotoxic effects of a carefully selected set of pharmacological compounds. Morphology, viability, and differentiation potential were investigated after exposing EBs to folic acid, all-trans-retinoic acid, dexamethasone, and valproic acid for 15 days. The results showed that the compounds differentially repressed cell growth, compromised morphology, and triggered apoptosis in the EBs. Further, transcriptomics was employed to compare subtle temporal changes between treated and untreated cultures. Gene ontology and pathway analysis revealed that dysregulation of a large number of genes strongly correlated with impaired neuroectoderm and cardiac mesoderm formation. This aberrant gene expression pattern was associated with several disorders of the brain like mental retardation, multiple sclerosis, stroke and of the heart like dilated cardiomyopathy, ventricular tachycardia, and ventricular arrhythmia. Lastly, these in vitro findings were validated using in ovo chick embryo model. Taken together, pharmacological compound or drug-induced defective EB development from hiPSCs could potentially be used as a suitable in vitro platform for embryotoxicity screening.