Single-cell analysis of human iPSC-derived midbrain organoids generated with triphasic Wnt modulation combined with a dynamic bioreactor

Human midbrain organoids (hMOs) derived from induced pluripotent stem cells offer a promising platform to model Parkinson’s disease (PD), but current systems often fall short in generating mature, regionally specified dopaminergic (DA) neurons that reflect disease-relevant vulnerabilities. Here, we present a refined differentiation strategy that combines tri-phasic WNT signaling modulation with dynamic bioreactor cultivation to enhance DA neuron yield, specification, and maturation within hMOs. This approach generated DA neurons with enriched substantia nigra-like identity (GIRK2⁺, ALDH1A1⁺), elevated dopamine release, and robust functional activity. Single cell transcriptomic profiling revealed upregulation of synaptic, metabolic, and neuroprotective pathways within the DA neuron cluster, while stress-related and pro-apoptotic programs were suppressed. Importantly, these hMOs exhibited selective vulnerability upon challenge with α-synuclein preformed fibrils (aSyn-PFFs), modeling PD-relevant neurodegeneration. Together, our findings establish a scalable, physiologically relevant platform for investigating PD mechanisms, therapeutic screening and advancing the development of cell replacement strategies.