Method Profile
A human cortical organoid system combines morphology, gene expression, cell composition and neuronal network measurements after brief exposure to the parent drug.
Study direct developmental-stage responses to acetaminophen and distinguish early molecular signals from later structural and functional measurements.
Cortical organoids derived from six independent human iPSC lines; individual readouts used different subsets of lines and organoids.
Vehicle-controlled exposure to 25, 50 or 100 μM acetaminophen for five days from differentiation day 21.
Human cortical organoids
Derived from six independent iPSC lines across the study.
Acetaminophen
Parent drug at 25, 50 and 100 μM, compared with vehicle.
Complementary measurements
Immunostaining, bulk and single-nucleus RNA sequencing, multi-electrode arrays and synaptic-marker analysis.
Analyze each measurement at its own biological sample level. The bulk RNA experiment included line and replicate structure; network activity was followed longitudinally.
Five-day parent-drug exposure beginning on differentiation day 21, followed by structural, transcriptomic, cell-composition and later network measurements.
Six independent iPSC lines overall; bulk RNA sequencing used 48 samples. Single-nucleus sequencing used eight samples from two lines and retained 55,665 nuclei. Multi-electrode recordings involved five lines and at least 14 organoids across three batches; synaptic analysis involved three lines and at least 19 organoids.
Vehicle control versus 25, 50 and 100 μM acetaminophen; a positive control was used for the apoptosis assay.
Bulk RNA sequencing found no differentially expressed genes at 25 μM, 10 at 50 μM and 16 at 100 μM. No significant later changes were detected in the reported cell-composition, single-nucleus expression, network or synaptic endpoints.
Researchers exposed human cortical organoids to acetaminophen for five days and followed structural, transcriptomic and network outcomes for several months. Small early transcriptional changes did not translate into detectable differences in later cellular composition or the measured functional endpoints.
