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.

在大型动物模型中构建与人类相关的多巴胺能（dopaminergic, DA）神经元，对转化神经科学与再生医学具有重要应用前景。本研究建立了一套稳健的实验方案，可从猪胚胎干细胞（porcine embryonic stem cells, pESCs）定向诱导尾侧腹侧中脑（caudal ventral midbrain, VM）命运，并生成具有功能的中脑多巴胺能（midbrain DA, mDA）神经元以及猪源中脑类器官（porcine midbrain-like organoids, pMLOs）。通过优化糖原合成激酶3（glycogen synthase kinase 3, GSK3）抑制策略与音猬因子（Sonic Hedgehog, SHH）信号通路，本研究明确了诱导猪源VM前体所需的物种特异性条件，其相较于人类细胞具有更高的敏感性阈值。单细胞与整体转录组测序分析显示，mDA神经元的谱系特化与功能成熟呈渐进式过程，且体外受精（in vitro fertilization, IVF）来源的pESCs其分化潜能优于孤雌激活（parthenogenetic activation, PA）来源的细胞。此外，三维分化体系可形成均一的神经上皮结构，并增强神经元的电生理活性与多巴胺释放能力。值得注意的是，多神经元簇中均检测到多巴胺能标志物的表达，这反映了高分辨率单细胞RNA测序所捕捉到的细胞身份获得的连续过程。相较于体内转基因猪模型，该平台可实现快速、可规模化且伦理上更易接受的中脑发育与DA神经元生物学研究。本研究结果证实，猪源类器官可作为在大型动物模型中模拟人类神经发育与疾病的高效工具。

实验整体设计：为探究中脑多巴胺能（mDA）神经元分化过程中的时序基因表达动态变化，本研究采用两种不同胚胎起源的猪胚胎干细胞（pESC）系——体外受精（IVF）来源与孤雌激活（PA）来源——进行整体转录组测序。研究人员在分化过程的四个代表性时间点收集两种细胞系的样本：第0天（未分化状态）、第16天（神经元早期特化阶段）、第28天（突触成熟阶段）以及第45天（晚期分化阶段）。每个时间点设置生物学重复样本，最终共计获得16份测序样品。本实验设计旨在捕捉随时间变化的转录组变化，并对比IVF与PA来源的mDA神经元的分化模式差异。研究通过相关性分析与基因本体论（gene ontology, GO）富集分析，评估细胞分化的时序进程与功能富集情况。本数据集可用于评估体外mDA神经元发育过程中，参与神经发生、突触形成、细胞应答以及谱系特异性分化的关键基因表达程序。