Effect of three triiodothyronine (T3) conditions on neuronal differentiation of human embryonic stem cell derived neural progenitors

Thyroid hormone (TH) is an important regulator of human brain development, and maternal TH imbalance is linked to adverse outcomes such as reduced IQ and decreased motorimpaired brain function in children. As part of the VHP4Safety initiative, we investigated the molecular effects of altered TH levels using the human neural progenitor test (hNPT). Neural progenitor cells were differentiated in vitro under no (0 nM), normal (4.1 nM), or high (410 nM) triiodothyronine (T3) conditions. RNA-Seq was used to assess gene expression, and a brain-specific molecular Adverse Outcome Pathway (AOP) network was applied to interpret transcriptomic changes alongside Gene Ontology (GO) enrichment analysis. No T3 exposure significantly altered gene expression in pathways related to neurogenesis, synaptogenesis, differentiation and metabolism, including pathways of BDNF signalling, proliferation, oligodendrocyte specification, and glycolysis. Key Events (KEs) such as reduced BDNF and impaired proliferation were enriched in the molecular AOP network. High T3 had minimal transcriptional effects. Our findings demonstrate that low T3 conditions affect key neurodevelopmental processes in vitro. This study illustrates the utility of integrating transcriptomics with molecular AOPs to provide a structured, mechanistic framework for evaluating the regulation of neuronal cell proliferation and differentiation in brain development. The framework may be useful for chemical risk assessment.