Next-generation sequencing of murine trophoblast-derived and pregnancy-associated exosome-enriched extracellular vesicle microRNAs

The role of extracellular vesicles (EVs), specifically exosomes, in intercellular communication likely plays a key role in placental orchestration of pregnancy and maternal immune sensing of the fetus. While murine models are powerful tools to study pregnancy and maternal-fetal immune interactions, in contrast to human placental exosomes, the content of murine placental and pregnancy exosomes remains largely understudied. Using a recently developed in vitro culture technique, murine trophoblast stem cells derived from B6 mice were differentiated into syncytial-like cells. EVs from the conditioned media, as well as from pregnant and non-pregnant sera, were enriched for exosomes. The RNA composition of these murine trophoblast-derived and pregnancy-associated exosome-enriched-EVs (ExoE-EVs) was determined using RNA-sequencing analysis and expression levels confirmed by qRT-PCR. Differentially abundant miRNAs were detected in syncytial differentiated ExoE-EVs, particularly from the X chromosome cluster (mmu-miR-322-3p, mmu-miR-322-5p, mmu-miR-503-5p, mmu-miR-542-3p, and mmu-miR-450a-5p). These were confirmed to be increased in pregnant mouse sera ExoE-EVs by qRT-PCR analysis. Interestingly, fifteen miRNAs were only present within the pregnancy-derived ExoE-EVs compared to non-pregnant controls. Mmu-miR-292-3p and mmu-miR-183-5p were noted to be some of the most abundant miRNAs in syncytial ExoE-EVs and were also present at higher levels in pregnant versus non-pregnant sera ExoE-EVs. The bioinformatics tool, MultiMir, was employed to query publicly available databases of predicted miRNA-target interactions. This analysis reveals that the X-chromosome miRNAs are predicted to target ubiquitin-mediated proteolysis and intracellular signaling pathways. Knowing the cargo of placental and pregnancy-specific ExoE-EVs as well as the predicted biological targets informs studies using murine models to examine not only maternal-fetal immune interactions but also the physiologic consequences of placental-maternal communication. Examination of EVs isolated from 1) conditioned media from in vitro mouse trophoblast cells cultures maintained as "STEM" using Cell Start and FGF4 (n=3), 2) conditioned media from in vitro mouse trophoblast cultures that have undergone syncytial-like differentiation (n=3), 3) non-pregnant B6 mouse sera (n=3); 4) pregnant B6 mouse sera (gestational day 14.5, n=3). EVs were isolated using ExoQuick-TC (1-2) or ExoQuick (3-4).

细胞外囊泡（extracellular vesicles, EVs），尤其是外泌体（exosomes），在细胞间通讯中所发挥的作用，可能在妊娠的胎盘调控以及母体对胎儿的免疫感知过程中扮演关键角色。尽管小鼠模型是研究妊娠与母胎免疫互作的有力工具，但相较于人类胎盘外泌体，小鼠胎盘及妊娠相关外泌体的内容物仍未得到充分研究。借助近期开发的体外培养技术，研究人员将源自B6小鼠的小鼠滋养层干细胞诱导分化为合胞体样细胞。从条件培养基、妊娠及非妊娠血清中分离得到的细胞外囊泡均经外泌体富集处理，得到外泌体富集囊泡（exosome-enriched-EVs, ExoE-EVs）。采用RNA测序（RNA-sequencing）分析测定了这些小鼠滋养层来源及妊娠相关ExoE-EVs的RNA组成，并通过实时定量聚合酶链反应（qRT-PCR）验证了其表达水平。在合胞体分化后的ExoE-EVs中检测到差异富集的微小RNA（microRNA, miRNA），尤其是来自X染色体簇的mmu-miR-322-3p、mmu-miR-322-5p、mmu-miR-503-5p、mmu-miR-542-3p及mmu-miR-450a-5p。经qRT-PCR分析证实，这些miRNA在妊娠小鼠血清的ExoE-EVs中表达上调。值得注意的是，与非妊娠对照组相比，有15种miRNA仅存在于妊娠来源的ExoE-EVs中。此外，mmu-miR-292-3p与mmu-miR-183-5p被发现是合胞体ExoE-EVs中丰度最高的miRNA之一，且在妊娠小鼠血清的ExoE-EVs中的表达水平亦显著高于非妊娠对照组。研究人员采用生物信息学工具MultiMir查询了公开的miRNA靶标互作预测数据库。分析结果显示，这些X染色体来源的miRNA预测靶向泛素介导的蛋白水解通路与细胞内信号通路。明确胎盘及妊娠特异性ExoE-EVs的内容物及其预测的生物学靶标，可为利用小鼠模型开展母胎免疫互作及胎盘-母体通讯的生理效应相关研究提供理论支撑。本研究共采集四类样本：1）采用Cell Start基质与FGF4维持于干细胞态的体外小鼠滋养层细胞条件培养基（n=3）；2）经合胞体样分化的体外小鼠滋养层细胞条件培养基（n=3）；3）非妊娠B6小鼠血清（n=3）；4）妊娠第14.5天的B6小鼠血清（n=3）。所有细胞外囊泡均采用ExoQuick-TC试剂盒（对应样本1、2）或ExoQuick试剂盒（对应样本3、4）分离得到。