The role of secreted exosomes during dopaminergic (DA) neuron differentiation is still unknown. To investigate the roles of exosomes in DA neuron fate specification, we profiled exosomal microRNAs (miRNAs) during DA neuron differentiation of epiblast-derived stem cells (EpiSCs). There were 26 miRNAs differentially expressed (relative fold >2, p < 0.05) in EpiSC-derived exosomes at 0, 2, 4, 6, 8, 10, 12, and 14 days of DA epiblast differentiation. Among them, 23 exosomic miRNAs were significantly increased, including miR-124, miR-132, miR-133b, miR-218, miR-9, miR-34b, miR-34c, and miR-135a2, while three exosomic miRNAs (miR-214, miR-7a, and miR-302b) were decreased, when compared with control samples. Bioinformatics analysis by DIANA-mirPath demonstrated that extracellular matrix-receptor interaction, signaling pathways regulating pluripotency of stem cells, FoxO signaling pathway, DA synapse, Wnt signaling pathway, GABAergic synapse, and neurotrophin signaling pathway were significantly enriched in DA differentiation-related miRNA signature (all p-values <0.012). Furthermore, messenger RNAs for nine DA neuronal markers tyrosine hydroxylase (TH), Nr4a2, Pitx3, Drd1a, Lmx1a, Lmx1b, Foxa1, Dmrt5, and Slc18a2 were significantly increased expressed over time in exosomes derived from differentiated EpiSCs. Interestingly, adding with exosomes derived from EpiSC induction experiment resulted in a twofold increase of TH-positive neurons production (35% vs. 17%, p < 0.01) during DA neuronal differentiation from mouse embryonic stem cells (ESCs). In summary, our results suggested exosomal miRNAs are potential regulators of DA neuron differentiation. More importantly, EpiSC-derived exosomes could promote the generation of DA neuron differentiation from ESCs.
Keywords: differentiation; dopaminergic neurons; exosome; microRNAs.