MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons

Direct neuronal conversion describes the process of generating induced neurons from somatic cells such as fibroblasts by overexpressing cell type-specific transcription factors. This was first achieved by expressing Brn2, Ascl1 and MytL1 in mouse fibroblasts, and was later achieved in human cells by 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 neuronal-specifying transcription factors and microRNAs and evaluated morphology and neuronal 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 neuronal programs increased and matured during conversion. We generated a heterogenous population of glutamatergic and GABAergic neurons. RNA sequencing of porcine induced neurons from fibroblast at three different timepoints during conversion. Three replicates were used for each timepoint.