Pregnancy is a unique situation, in which placenta-derived small extracellular vesicles (sEVs) may communicate with maternal and foetal tissues. While relevant to homoeostatic and pathological functions, the mechanisms underlying sEV entry and cargo handling in target cells remain largely unknown. Using fluorescently or luminescently labelled sEVs, derived from primary human placental trophoblasts or from a placental cell line, we interrogated the endocytic pathways used by these sEVs to enter relevant target cells, including the neighbouring primary placental fibroblasts and human uterine microvascular endothelial cells. We found that trophoblastic sEVs can enter target cells, where they retain biological activity. Importantly, using a broad series of pharmacological inhibitors and siRNA-dependent silencing approaches, we showed that trophoblastic sEVs enter target cells using macropinocytosis and clathrin-mediated endocytosis pathways, but not caveolin-dependent endocytosis. Tracking their intracellular course, we localized the sEVs to early endosomes, late endosomes, and lysosomes. Finally, we used coimmunoprecipitation to demonstrate the association of the sEV microRNA (miRNA) with the P-body proteins AGO2 and GW182. Together, our data systematically detail endocytic pathways used by placental sEVs to enter relevant fibroblastic and endothelial target cells, and provide support for “endocytic escape” of sEV miRNA to P-bodies, a key site for cytoplasmic RNA regulation.

怀孕是一种独特的情况，胎盘来源的小细胞外囊泡（sEVs）可能与母体和胎儿组织通讯。尽管与稳态和病理功能有关，但sEV进入和靶细胞中货物处理的基本机制仍然未知。使用荧光或发光标记的sEV衍生自原代人胎盘滋养细胞或胎盘细胞系，我们询问了这些sEV进入相关靶细胞的内吞途径，包括邻近的原代胎盘成纤维细胞和人子宫微血管内皮细胞。我们发现滋养细胞sEVs可以进入靶细胞，并在其中保留生物活性。重要的是，使用各种药理抑制剂和siRNA依赖性沉默方法，我们显示，滋养层sEV使用巨胞饮和网格蛋白介导的内吞途径进入靶细胞，但不依赖小窝蛋白依赖性内吞。跟踪它们的细胞内过程，我们将sEVs定位于早期内体，晚期内体和溶酶体。最后，我们使用免疫共沉淀来证明sEV microRNA（miRNA）与P体蛋白AGO2和GW182的关联。总之，我们的数据系统地详细说明了胎盘sEV进入相关的成纤维细胞和内皮靶细胞所使用的内吞途径，并为sEV miRNA向P体（细胞质RNA调控的关键位点）“内逃”提供了支持。我们将sEV定位于早期内体，晚期内体和溶酶体。最后，我们使用免疫共沉淀来证明sEV microRNA（miRNA）与P体蛋白AGO2和GW182的关联。总之，我们的数据系统地详细说明了胎盘sEV进入相关的成纤维细胞和内皮靶细胞所使用的内吞途径，并为sEV miRNA向P体（细胞质RNA调控的关键位点）“内逃”提供了支持。我们将sEV定位于早期内体，晚期内体和溶酶体。最后，我们使用免疫共沉淀来证明sEV microRNA（miRNA）与P体蛋白AGO2和GW182的关联。总之，我们的数据系统地详细说明了胎盘sEV进入相关的成纤维细胞和内皮靶细胞所使用的内吞途径，并为sEV miRNA向P体（细胞质RNA调控的关键位点）“内逃”提供了支持。