Extracellular vesicles and their ncRNA cargo as markers of trophoblast injury

 描述(申请人提供)：在整个人类怀孕期间，胎盘对于胚胎发育、胎儿生长和组织分化是不可或缺的。胎盘还可以保护胎儿免受各种侮辱，同时保护孕妇的健康。胎盘功能障碍通常与妊娠并发症有关，这些并发症挑战母体生理(如先兆子痫)和胎儿发育(如胎儿死亡或胎儿生长受限)，或导致早产。在胎盘内，滋养层构成最外层，直接沐浴在母体血液中，因此处于调节母胎气体交换、营养输送、废物清除和激素产生的位置，忠实地平衡胎儿需求和母体供应。滋养细胞损伤在功能不全的胎盘中很常见，可能会破坏微妙的母婴平衡，导致临床疾病，并给健康留下终身的印记。围产期医学的一个根本挑战来自于我们在整个怀孕期间实时诊断胎盘疾病的有限能力。然而，我们和其他人最近的发现：(A)胎盘滋养层细胞向母体循环释放独特的微囊泡和纳米囊泡，(B)这些囊泡含有滋养层细胞特有的非编码RNA货物，为评估滋养层细胞的健康创造了新的机会。这些囊泡在整个妊娠期间由滋养层细胞主动释放，从而成为静脉穿刺法可获得的滋养层细胞的自然活检，可以实时提供关于滋养层健康的信息。我们在Magee-Women研究所建立的围产期生物学小组包括围产期医学和胎盘病理学、发育和分子生物学以及生物信息学方面的专业知识。受这些最新进展的启发，我们与一群经验丰富的生物工程师合作，其中包括来自卡内基梅隆大学、麻省理工学院和宾夕法尼亚州立大学的专家，他们在基于生物物理学的囊泡分析方面拥有独特的技能，包括微流体、纳米机械、微/纳米制造和使用声波镊子进行囊泡分类。我们的新的跨学科小组将使用集成的分子和生物物理方法来直接评估从母体血浆中提取的滋养层细胞外小泡的使用情况，以实时揭示滋养层细胞的健康状况，并将其作为一种可能在整个怀孕期间使用的技术。我们的方法是全面的，以在外体纳米囊以及微泡和凋亡小体中分析的miRNAs以及lncRNAs和CircRNAs为中心。由于每个小泡都具有独特的双分子和生物物理特征，我们将部署我们基于机器学习的培训和测试管道，将这些不同的信号信息集成到一个创新的诊断工具中。最后，我们部署了负担得起的声镊子技术来分选滋养层小泡，这将有助于将我们的进步转化为一种适用于小血量的新的“芯片实验室”胎盘诊断技术。这项技术不仅可以指示滋养层细胞的病理，而且有可能识别哪些人可能从干预中受益，并监测治疗的成功。

 DESCRIPTION (provided by applicant): Throughout human pregnancy, the placenta is indispensable for embryonic development, fetal growth and tissue differentiation. The placenta also protects the fetus against diverse insults, while preserving maternal health. Placental dysfunction is commonly implicated in complications of pregnancy that challenge maternal physiology (e.g., preeclampsia) and fetal development (e.g., fetal death or fetal growth restriction) or that leads to preterm birth. Within the placenta, the trophoblast constitutes the outermost layer, which is directly bathed in maternal blood and therefore positioned to regulate maternal-fetal gas exchange, nutrient delivery, waste removal and the production of hormones, faithfully balancing fetal needs and maternal supply. Trophoblast damage, which is common in dysfunctional placentas, may interrupt the delicate maternal-fetal balance, cause clinical disease, and leave a lifelong mark on health. A fundamental challenge in perinatal medicine arises from our limited ability to diagnose placental disorders in real time and throughout pregnancy. However, the recent discovery, by ourselves and others, that (a) placental trophoblasts release distinctive micro- and nanovesicles into the maternal circulation and (b) these vesicles contain trophoblast-specific non-coding RNA cargo, created a new opportunity for assessing trophoblast health. These vesicles are actively released by trophoblasts throughout pregnancy, and thus serve as a venipuncture-accessible "natural biopsy" of trophoblasts, which can furnish information on trophoblast health in real time. Our established perinatal biology group at Magee- Womens Research Institute includes expertise in perinatal medicine and placental pathology, developmental and molecular biology, and bioinformatics. Inspired by these recent advances, we have partnered with an experienced group of bioengineers that includes experts from Carnegie Mellon University, MIT, and Penn State University, with unique skills in biophysics-based vesicle analytics, including microfluidics, nanomechanics, micro/nano fabrication and vesicle sorting using acoustic tweezers. Together, our new transdisciplinary group will use integrated molecular and biophysical methodologies to directly assess the use of trophoblast-derived extracellular vesicles from maternal plasma as revelatory of trophoblast health in real time and as a technique that may be employed throughout pregnancy. Our approach is comprehensive, centering on miRNAs as well as lncRNAs and circRNAs, analyzed in exosome nanovesicles, as well as microvesicles and apoptotic bodies. As each vesicle features a unique bimolecular and biophysical signature, we will deploy our machine learning- based training and testing pipeline to informatively integrate these distinct signals into an innovative diagnostic tool. Lastly, our deployment of affordable acoustic tweezers technology to sort trophoblastic vesicles will facilitate the translation of our advances into a new "lab on a chip" placental diagnostic technology, suitable for small blood volumes. This technology may not only denote trophoblast pathology, but has potential to identify those who may benefit from intervention and to monitor therapeutic success.