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.

中文翻译：

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在多巴胺能神经元分化过程中，干细胞衍生的外来体中microRNA轮廓和Wnt信号的差异表达。

尚不清楚分泌的外泌体在多巴胺能（DA）神经元分化过程中的作用。为了研究外泌体在DA神经元命运规范中的作用，我们在上皮来源干细胞（EpiSCs）的DA神经元分化过程中，对外泌体微RNA（miRNA）进行了分析。有26个miRNA差异表达（相对倍数> 2，p <0.05）在DA表皮细胞分化的0、2、4、6、8、10、12和14天时来自EpiSC的外泌体。其中23种外泌体miRNA显着增加，包括miR-124，miR-132，miR-133b，miR-218，miR-9，miR-34b，miR-34c和miR-135a2，而三个外泌体miRNA（miR与对照样品相比，-214，miR-7a和miR-302b）降低。通过DIANA-mirPath进行的生物信息学分析表明，细胞外基质-受体相互作用，调节干细胞多能性的信号通路，FoxO信号通路，DA突触，Wnt信号通路，GABA能突触和Neurotrophin信号通路显着丰富了DA分化相关的miRNA信号。 （全部p-值<0.012）。此外，九种DA神经元标记酪氨酸羟化酶（TH），Nr4a2，Pitx3，Drd1a，Lmx1a，Lmx1b，Foxa1，Dmrt5和Slc18a2的信使RNA随着时间的推移在分化的EpiSC衍生的外泌体中表达明显增加。有趣的是， 在小鼠胚胎干细胞（ESC）分化为DA的神经元过程中，添加EpiSC诱导实验衍生的外来体导致TH阳性神经元产量增加了两倍（35％比17％，p <0.01）。总之，我们的结果表明，外泌体miRNA是DA神经元分化的潜在调节剂。更重要的是，EpiSC衍生的外来体可促进ESC产生DA神经元分化。