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

ABSTRACT: 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.

ORGANISM(S): Sus scrofa

PROVIDER: GSE296089 | GEO | 2025/09/12

REPOSITORIES: GEO

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Project description:Induced pluripotent stem cells (iPSCs) hold great promise for in vitro disease modeling and cell replacement therapy for Parkinson’s disease (PD). Both applications crucially require an in-depth profiling of the disease-relevant, iPSC-derived cell type. Midbrain dopaminergic (mDA) neurons derived from pluripotent stem cells are of substantial interest because of their instrumental value for PD therapy. IPSC-derived mDA neuron-like cells have been generated, however, detailed genetic and epigenetic characterization of strictly purified in vitro generated DA neurons has so far lagged behind. We generated mouse Pitx3gfp/+ iPSC-derived DA neurons that, after fluorescent activated cell sorting (FACS) allowed comprehensive comparison to mesodiencephalic dopaminergic (mdDA) neurons from Fac-sorted Pitx3gfp/+ ventral midbrains. We performed detailed analysis of global gene expression and genome-scale DNA methylation of CpG islands (CGIs) by reduced representation bisulfite sequencing. The reprogramming pathway from fibroblasts to iPSCs left parental cell footprints for both gene expression and DNA methylation. However, most gene expression patterns of iPSC-derived DA neurons closely resembled that of primary mdDA neurons with the strongest correlations for mdDA specific genes. Also, for DNA methylation patterns, high similarities were found for the vast majority of CGIs when comparing primary mdDA neurons with iPSC-derived DA neurons. Additionally, we found de novo DNA methylation during in vitro differentiation for hundreds of genes specifically in lineage-committed neural precursors that persisted in iPSC-derived DA neurons. Our study provides novel detailed characteristics of iPSC-derived DA neurons in comparison to the primary cell type. These findings add important information to our knowledge about these biomedically highly valuable, in vitro generated neurons. Microarray expression study comparing 3 samples of facs-sorted, Pitx3-gfp positive cells from each experimental group to a common reference consisting of adult mouse midbrain RNA. Each sample was analysed in normal and opposite dye orientation.

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

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