Engineering functional ventral midbrain dopaminergic neurons in human organoids through WNT modulation and bioreactor culture.

Human midbrain organoids (hMOs) derived from induced pluripotent stem cells provide a powerful system to model disorders involving dopamine (DA) dysfunction, including Parkinson's disease (PD) and neuropsychiatric conditions. However, current differentiation protocols still fall short in recapitulating early specification, substantia nigra pars compacta (SNpc)-like identity, and the functional maturation of vulnerable DA neurons. Here, we established a differentiation strategy that combines tri-phasic WNT modulation with dynamic bioreactor culture to generate hMOs enriched in SNpc-like DA neurons. This approach significantly increases the yield of TH⁺/GIRK2⁺ and TH⁺/ALDH1A1⁺ DA neurons and promotes enhanced synaptic maturation, robust electrophysiological activity, and elevated DA release. Single-cell transcriptomics revealed that this strategy drives the emergence of SOX6+/GIRK2+ SNpc-like neurons, accompanied by upregulation of synaptic, metabolic, and maturation programs, alongside reduced cell stress and apoptotic signaling. Importantly, hMOs demonstrated vulnerability upon exposure to α-synuclein preformed fibrils, resulting in aggregate formation and DA neuron degeneration, supporting their use as a human model of PD-relevant pathology. Overall, this system provides a scalable and physiologically relevant approach to investigate molecular mechanisms underlying neurodegeneration and DA-related disorders.