P-Exo-CyTOF: Opportunity to Assess Human Placental Function in Real-Time

 描述（由申请人提供）：人类发育取决于胎盘的发育和功能。这个器官连接母亲和胎儿，提供氧气和营养物质的运输，并作为怀孕的主要调节器。了解人类胎盘的发育和功能一直具有挑战性，因为胎盘的结构和发育在哺乳动物物种中并不高度保守，并且对正在进行的人类妊娠的纵向分析受到实际和伦理考虑的限制。胎盘释放各种小的膜结合颗粒进入母体循环。这些胎盘包提供了评估胎盘功能状态的机会。该项目旨在开发一种新的工具来监测这些包装类型中的一种类型，即外泌体，因为货物反映了起源组织的生理状态。该方法是将CyTOF（串联流式细胞术和质谱技术）的能力从单细胞水平的询问扩展到单囊泡水平，以分析来自母体循环的胎盘特异性外泌体，从而揭示人类胎盘的功能状态。这将通过以下目标来实现。(1)开发并验证一种新的工具（P-Exo-CyTOF），使用CyTOF平台鉴定人类胎盘特异性外泌体。将开发和优化创新的基于金属的囊泡标记、胎盘外泌体的特异性金属标记抗体组和新型计算工作流程，以通过CyTOF检测胎盘来源的外泌体。来源于在各种氧分压下培养的人滋养层细胞系以及来自血压正常和先兆子痫妊娠的母体血浆的外泌体将用于验证和测试P-Exo-CyTOF在原理实验证明中的灵敏度、特异性和效用。(2)确定外泌体标志物，反映妊娠和功能状态的人胎盘。使用半定量质谱法，将确定妊娠期间和先兆子痫背景下的人胎盘外泌体的蛋白质组。这些研究将为代表胎盘细胞状态的综合功能性P-Exo-CyTOF抗体组的组成提供信息。该提案不仅将提高CyTOF在细胞外囊泡评估方面的能力，而且将提供在正在进行的妊娠期间实时安全研究和监测人类胎盘的能力。

 DESCRIPTION (provided by applicant): Human development depends on the development and function of the placenta. This organ connects the mother to her fetus providing transport of oxygen and nutrients and serves as a master regulator of pregnancy. Understanding human placenta development and function has been challenging in that placental structure and development is not highly conserved across mammalian species and longitudinal analysis of on-going human pregnancies is limited by practical and ethical considerations. The placenta releases a variety of small membrane bound particles into the maternal circulation. These placental packages provide an opportunity to assess the functional state of the placenta. This project seeks to develop a novel tool to monitor one type of these package types, exosomes, as the cargo reflects the physiologic state of the tissue of origin. The approach is to extend the capabilities of CyTOF (a tandem flow cytometry and mass spectroscopy technology) from the single-cell level of interrogation to the single-vesicle level to analyze placental-specific exosomes from maternal circulation to reveal the functional state of the human placenta. This will be done through the following aims. (1) Develop and validate a novel tool (P-Exo-CyTOF) to identify human placental-specific exosomes using the CyTOF platform. Innovative metal-based vesicle labeling, specific metal-tagged antibody panels for placental exosomes and novel computational workflows will be developed and optimized to enable the detection of placenta-derived exosomes by CyTOF. Exosomes derived from human trophoblast cell lines cultured in various oxygen tensions as well as maternal plasma from normotensive and pre-eclamptic pregnancies will be used to validate and test the sensitivity, specificity and utility of P-Exo-CyTOF in proof of principle experiments. (2) Determine exosome markers that reflect the gestational and functional state of the human placenta. Using semi-quantitative mass spectrometry, the proteome of human placental exosomes over gestation and in the context of preeclampsia will be determined. These studies will inform the composition of comprehensive functional P-Exo-CyTOF antibody panels representative of placental cellular states. Not only will this proposal advance the capabilities of CyTOF in terms of extracellular vesicle assessment but will provide the ability to safely study and monitor the human placenta in real-time during an on-going pregnancy.