3D-generation of high-purity midbrain dopaminergic progenitors and lineage-guided refinement of grafts supports Parkinson's disease cell therapy.

The low in vivo yield of midbrain dopaminergic (mDA) neurons and uncertain lineage fates of donor cells following transplantation impede clinical application of human pluripotent stem cell (hPSC)-based cell therapy for Parkinson's disease (PD). We developed a three-dimensional (3D) differentiation method, SphereDiff, to generate high-purity mDA progenitors (mDAPs), leading to a significant enrichment of mDA neurons post transplantation. Grafted mDA neurons fully restored dopamine levels and corrected motor deficits in PD model mice. Single-cell spatial transcriptomics revealed a patterned distribution of mDA neuron subtypes and glial cells. Using cross-transplantation single-cell split barcoding (TX-SISBAR), we elucidated the clonal lineage fates of donor cells post transplantation, revealing the mDA neuron and astrocyte fates of mDAPs and glutamatergic neuron fates of diencephalic progenitors. Leveraging these lineage insights, we further refined SphereDiff and eliminated off-target lineage cells. Producing high in vivo efficacy, lineage-defined donor cells supports safer and more effective PD cell therapy in regenerative medicine.