Cold Spring Harbor Laboratory

Human midbrain organoids (hMOs) derived from induced pluripotent stem cells represent a promising model for Parkinson’s disease (PD). However, current protocols often fall short in producing mature, regionally specified dopaminergic (DA) neurons that faithfully recapitulate disease-relevant vulnerabilities. Here, we present a differentiation strategy combining tri-phasic WNT signaling modulation with dynamic bioreactor culture to enhance DA neuron yield, regional identity, and functional maturation within hMOs. This approach generated DA neurons with enriched substantia nigra-like features, increased dopamine release, and robust electrophysiological activity. Single-cell transcriptomic analysis revealed upregulation of synaptic, metabolic, and neuroprotective pathways, alongside reduced expression of stress and pro-apoptotic signatures in DA neurons. Importantly, these hMOs demonstrated vulnerability upon exposure to α-synuclein preformed fibrils and proteinase K-resistant aggregates, effectively modeling PD-associated pathology. Altogether, our platform offers a scalable and physiologically relevant system for investigating PD mechanisms, enabling therapeutic screening, and supporting the advancement of cell replacement strategies.