Developmentally Guided Differentiation of Ventral Midbrain Dopaminergic Cells Validated in Proteopathy‐Based Parkinson's Disease Models

Parkinson's disease (PD) involves progressive degeneration of nigrostriatal ventral midbrain dopaminergic (vmDA) neurons and Lewy body proteopathy, necessitating scalable, lineage-faithful donor cells. However, variability in human pluripotent stem cell (hPSC) differentiation and incomplete ventral midbrain identity limit translational use. Here, we establish a developmentally guided differentiation protocol that reproducibly specifies EN1-positive vmDA progenitors through temporally optimized SHH, WNT/β-catenin, and FGF8 signaling. The resulting progenitors mature into vmDA neurons exhibiting stimulus-dependent dopamine release and maturation-associated pacemaking activity. Progenitors can be cryopreserved at a defined stage while retaining viability and neurogenic capacity. Functional validation was performed using complementary proteopathy-based PD models. In vitro, optogenetic induction of α-synuclein aggregation (OASIS) triggered robust aggregate formation and selective degeneration of TH-positive vmDA neurons in both two-dimensional cultures and three-dimensional neurospheroids. In vivo, we established a conditional β23 proteopathy-based PD mouse model exhibiting progressive nigrostriatal degeneration and motor impairment. Transplantation of hESC-derived vmDA progenitors into advanced-stage PD mice led to graft survival, differentiation into vmDA neurons, and significant motor improvement. Together, these findings define a robust vmDA differentiation framework validated across proteopathy-based disease modeling and transplantation contexts, supporting its application in PD modeling and cell therapy.