In vitro derivation of midbrain dopaminergic neurons from porcine embryonic stem cells in multi-dimensional conditions

Background: . Porcine pluripotent stem cells hold significant promise as a large-animal model for human neurodevelopment and disease modeling. However, efficient protocols for their directed differentiation into midbrain dopaminergic (mDA) neurons and the generation of 3D midbrain-like organoids remain limited. This study aimed to establish species-optimized conditions for the derivation of functional mDA neurons from porcine embryonic stem cells (pESCs) under both two- and three-dimensional environments.

Methods: . In vitro fertilization (IVF)- and parthenogenetic activation (PA)-derived pESCs were subjected to neural induction using stepwise exposure to dual SMAD inhibition, SHH, CHIR99021, and FGF8. For monolayer culture, adherent monolayers were differentiated on Matrigel- or poly-L-ornithine/laminin I/fibronectin-coated surfaces. For 3D culture, porcine midbrain-like organoids (pMLOs) were formed under low-adhesion conditions. Functional and molecular characterization was performed via immunofluorescence, patch-clamp electrophysiology, microelectrode array (MEA) recordings, dopamine ELISA, and RNA Sequencing.

Results: . Porcine embryonic stem cells required higher SHH and GSK3 inhibition thresholds to efficiently induce FOXA2⁺ ventral midbrain progenitors, revealing a species-specific divergence from human protocols. IVF-derived pESCs showed markedly enhanced dopaminergic differentiation and functional maturation compared to PA-derived pESCs. Notably, in 3D culture, neuroepithelial structures emerged as early as day 5, and functionally mature mDA neurons-confirmed by TH expression, dopamine release, and spontaneous synaptic activity-were detected by day 28. This timeline represents an accelerated maturation relative to comparable human mDA differentiation systems, where functional properties typically arise after day 35. Single-cell RNA Sequencing further delineated dynamic dopaminergic lineage trajectories and revealed porcine-specific gene expression patterns associated with early mDA identity acquisition.

Conclusions: . This study presents the first robust platform for generating functionally validated mDA neurons and midbrain-like organoids from porcine stem cells. The findings highlight species-specific signaling dynamics and establish porcine in vitro models as scalable, translational systems for investigating neurodevelopment and disease mechanisms.