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

 描述(申请人提供)：人类的发育取决于胎盘的发育和功能。这个器官将母亲和胎儿连接起来，提供氧气和营养的运输，是怀孕的主要调节器。了解人类胎盘的发育和功能一直是具有挑战性的，因为胎盘的结构和发育在哺乳动物物种中并不是高度保守的，而且对正在进行的人类怀孕的纵向分析受到实际和伦理考虑的限制。胎盘释放出各种微小的膜结合颗粒进入母体循环。这些胎盘包装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裹类型中的一种--外切体，因为货物反映了起源组织的生理状态。该方法是将CyTOF(一种串联的流式细胞仪和质谱仪技术)的能力从单细胞水平的询问扩展到单囊水平，以分析母体循环中的胎盘特异性外体，以揭示人类胎盘的功能状态。这将通过以下目标来实现。(1)开发和验证一种新的工具(P-Exo-CyTOF)，以利用CyTOF平台识别人胎盘特异的外切体。将开发和优化创新的基于金属的囊泡标记、用于胎盘外切体的特定金属标记抗体面板和新的计算工作流程，以实现细胞飞行时间对胎盘来源的外切体的检测。从不同氧分压下培养的人滋养层细胞系以及正常血压和先兆子痫孕妇的母体血浆中提取的外体将用于验证和测试P-Exo-CyTOF在原理实验中的敏感性、特异性和实用性。(2)确定能反映人胎盘妊娠状态和功能状态的外体标志物。利用半定量质谱仪，将确定妊娠期间和先兆子痫情况下人胎盘外切体的蛋白质组。这些研究将提供代表胎盘细胞状态的综合功能P-Exo-CyTOF抗体小组的组成。这项建议不仅将提高细胞外小泡评估方面的细胞外小泡评估能力，而且将提供在持续怀孕期间安全地研究和实时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.