Therapeutic Potential of iPSC-Derived TH⁺/FOXA2⁺ Neuronal Extracellular Vesicles in Parkinson’s Disease Revealed by Organoid and Rodent Models
Abstract
Background Parkinson’s disease (PD) is characterized by the progressive loss of midbrain dopaminergic (DA) neurons and aberrant α-synuclein (α-syn) aggregation, yet disease-modifying therapies that simultaneously retard neurodegeneration and foster regeneration remain lacking. Methods In this study, we isolated extracellular vesicles (TH⁺/FOXA2⁺ cell-derived EVs) from TH⁺/FOXA2⁺ differentiated neural cells, a distinct midbrain floor-plate-derived neural cell population that co-expresses the floor-plate transcription factor FOXA2 together with TH and should therefore not be equated with conventional mature midbrain dopaminergic neurons, and systematically evaluated their therapeutic potential across three complementary models: A53T transgenic mice, 6-OHDA-lesioned-rats, and 6-OHDA-treated human midbrain organoids. Integrative transcriptomic and single-cell-sequencing analyses, together with toxicological evaluations in rodents and non-human primates, were performed to investigate the underlying mechanisms and safety profile. These TH⁺/FOXA2⁺ cells were generated using a fully suspension-based, matrix-free, and chemically defined differentiation system devoid of N2/B27 supplements and fetal bovine serum (FBS). This approach offers a simple, controllable, low-cost, and scalable platform with high batch-to-batch consistency for EV manufacturing. Throughout this study, the term TH⁺/FOXA2⁺ cells refers to this distinct TH⁺/FOXA2⁺ cell population and should not be interpreted as conventional terminally differentiated dopaminergic neurons. Results Intranasal administration of TH⁺/FOXA2⁺ cell-derived EVs in A53T mice and 6-OHDA-lesioned rats significantly ameliorated motor deficits, increased nigral tyrosine hydroxylase (TH)-positive-neuron counts, reduced α-syn-aggregation, and suppressed gliosis. In human midbrain organoids, TH⁺/FOXA2⁺ cell-derived EVs preserved DA neuronal morphology and reduced GFAP and α-syn expression. Notably, when EVs were administered simultaneously with 6-OHDA modeling rather than after modeling was completed, the protective effect was stronger. Multi-omics-analysis revealed that TH⁺/FOXA2⁺ cell-derived EVs reversed pathological signatures of interferon-responsive-microglia and oxidative stress-adapted astrocytes, while restoring WNT and non-canonical-WNT mediated intercellular communications. Safety evaluations showed no discernible organ toxicity. Conclusions TH⁺/FOXA2⁺ cell-derived EVs exert neuroprotective effects across multiple PD models by modulating specific glial subpopulations and reinstating development-associated signaling pathways. Together with a fully suspension, matrix-free and N2/B27-free differentiation process that enables low-cost, scalable and highly reproducible manufacturing, TH⁺/FOXA2⁺ cell-derived EVs represent a promising therapeutic candidate for Parkinson’s disease.-