In vitro Derivation of Midbrain Dopaminergic Neurons from Porcine Embryonic Stem Cells in Multi-dimensional Conditions [scRNA-seq]

The generation of human-relevant dopaminergic (DA) neurons in large-animal models holds significant promises for translational neuroscience and regenerative medicine. Here, we establish a robust protocol to induce caudal ventral midbrain (VM) fate and generate functional midbrain DA (mDA) neurons and porcine midbrain-like organoids (pMLOs) from porcine embryonic stem cells (pESCs). Optimization of GSK3 inhibition and SHH signaling revealed species-specific requirements for inducing porcine VM progenitors, including a higher sensitivity threshold compared to human cells. Single-cell and bulk transcriptomic analyses demonstrated progressive lineage specification and functional maturation of mDA neurons, with IVF-derived pESCs showing superior differentiation potential over PA-derived cells. Moreover, 3D differentiation led to the formation of uniform neuroepithelial structures and enhanced electrophysiological activity and dopamine release. Notably, dopaminergic markers emerged across multiple neuronal clusters, reflecting a continuum of identity acquisition captured by high-resolution single-cell RNA sequencing. Compared to in vivo transgenic pig models, this platform enables rapid, scalable, and ethically favorable investigation of midbrain development and DA neuron biology. These findings position pig-derived organoids as a powerful tool for modeling human neurodevelopment and disease in a large-animal context. Single-cell RNA sequencing was performed on pigMLOs at days 11, 28, and 45 of differentiation to characterize temporal changes in cellular composition and transcriptional dynamics. After alignment to the Sus scrofa Sscrofa11.1 genome and downstream processing, 103,310 high-quality cells were obtained and visualized via UMAP.