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.