ABSTRACT: Acetaminophen (APAP) is the most widely used analgesic during pregnancy, yet its effects on prenatal human brain development remain incompletely understood. Epidemiological studies have reported inconsistent associations between prenatal APAP exposure and later neurodevelopmental outcomes, underscoring the need for mechanistic evaluation in human-relevant developmental models. Here, we examined how APAP influences early cortical development using induced pluripotent stem cell-derived cortical organoids (COs) generated from six independent lines. Organoids were exposed to physiologically relevant APAP concentrations (25, 50, and 100 μM) for 5 days beginning at day 21 of differentiation, corresponding to late first-trimester cortical development. We assessed organoid growth, apoptosis, differentiation, synaptic maturation, transcriptomic profiles using bulk and single-nucleus RNA sequencing (snRNA-seq), and functional network activity using multielectrode array recordings up to 4 months. APAP exposure did not significantly affect organoid size, cytoarchitecture, or viability. Neuronal and progenitor cell proportions, as well as synaptic puncta density, were not significantly unchanged. Bulk RNA-seq revealed subtle transcriptional changes only at the highest dose (16 differentially expressed genes at 100 μM), enriched for neurodevelopmental pathways. In contrast, snRNA-seq at 3 months revealed no changes in cell type composition or gene expression. Consistent with these findings, electrophysiological measures including firing rate, burst frequency, and network synchrony, were indistinguishable from controls. Together, these results indicate that exposure to therapeutic APAP concentrations during a critical window of early cortical development produces minimal molecular perturbations without detectable changes or effects within the sensitivity of our assay. This study provides mechanistic evidence indicating that, in this human organoid model, recommended APAP exposure is not associated with detectable disruptions in pathways implicated in brain development.
