In vitro Derivation of Midbrain Dopaminergic Neurons from Porcine Embryonic Stem Cells in Multi-dimensional Conditions [bulkRNA-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. Overall design: To investigate temporal gene expression dynamics during the differentiation of midbrain dopaminergic (mDA) neurons, bulk RNA sequencing was conducted using two porcine embryonic stem cell (pESC) lines derived from different embryonic origins: in vitro fertilization (IVF) and parthenogenetic activation (PA). Cells from both lines were collected at four representative time points during differentiation: day 0 (undifferentiated), day 16 (early neuronal specification), day 28 (synaptic maturation), and day 45 (late-stage differentiation). Each time point included biological duplicates, resulting in a total of 16 samples.  The experimental design aimed to capture transcriptomic changes over time and to compare differentiation patterns between IVF- and PA-derived mDA neurons. Correlation and gene ontology analyses were performed to assess temporal progression and functional enrichment. The dataset enables evaluation of key gene expression programs involved in neurogenesis, synaptogenesis, cellular responses, and lineage-specific differentiation during in vitro mDA neuron development.