Direct neuronal conversion describes the process of generating induced neurons from somatic cells such as fibroblasts by overexpressing cell type-specific transcription factors, microRNAs or by culturing in the presence of small molecules. This was first achieved by expressing Brn2, Ascl1 and Myt1L in mouse fibroblasts, and was later achieved in human cells by the inclusion of additional factors such as NeuroD1. Here, we present the first protocol for directly converting porcine fibroblasts into induced neurons. We used lentivirus-mediated delivery of previously identified neuron-specifying transcription factors and microRNAs and evaluated morphology and neuron marker expression after ten days of conversion. We found that Ascl1 and microRNAs, miR-9/9* and miR-124 together generated more neuronal cells than other conditions tested. The porcine induced neurons expressed common mature markers such as MAP2 and Synaptophysin after four weeks of conversion. Transcriptomic analysis revealed that fibroblast-specific signatures were silenced early in the conversion process, while the neuron-specific genes became more abundant during conversion. We generated a heterogeneous population of glutamatergic and GABAergic neurons.

直接神经元转换描述了通过过度表达细胞类型特异性转录因子，microRNA或在小分子存在下培养，从体细胞（例如成纤维细胞）生成诱导神经元的过程。这首先是通过在小鼠成纤维细胞中表达Brn2，Ascl1和Myt1L来实现的，后来通过包含其他因子（例如NeuroD1）而在人细胞中实现了。在这里，我们提出了直接将猪成纤维细胞转化为诱导神经元的第一个协议。我们使用慢病毒介导的先前鉴定的神经元特异性转录因子和microRNA介导的传递，并在转换十天后评估了形态和神经元标志物的表达。我们发现Ascl1和microRNA，miR-9 / 9 *和miR-124在一起产生的神经元细胞比其他测试条件要多。转化后四周，猪诱导的神经元表达了常见的成熟标志物，例如MAP2和突触素。转录组学分析显示，成纤维细胞特异的信号在转化过程早期被沉默，而神经元特异的基因在转化过程中变得更加丰富。我们生成了谷氨酸能和GABA能神经元的异质群体。