Perez Fernandez, R.; Siekmann, M. T.; Gasparotto, M.; Wallhorn, L.; Artioli, A.; Kubitschke, M.; Guida, C.; Jabali, A.; Hoffrichter, A.; Koch, P.; Masseck, O. A.; Ladewig, J.

Neuromodulatory signaling is classically associated with mature neural circuits, yet serotonergic projections reach the developing cortex prior to circuit formation, suggesting a role in early human cortical development. Here, we establish a human raphe-cortical assembloid platform by generating iPSC-derived hindbrain-patterned raphe organoids that produce serotonergic neurons and form projections into fused cortical organoids, exhibiting endogenous serotonin release within developing cortical tissue. Using this system, we find that serotonergic innervation is associated with a shift toward progenitor-enriched states, accompanied by increased proliferative activity, activation of developmental transcriptional programs, and predicted signaling interactions targeting cortical progenitors and neurons. Consistent with these findings, cortical regions receiving serotonergic projections exhibit increased mitotic activity, and pharmacological modulation demonstrates selective proliferative responses in basal progenitor populations, consistent with observations in human fetal tissue. Together, this system provides a framework to investigate how early neuromodulatory input shapes human cortical development and developmental vulnerability.