Placental cell-and size-specific extracellular vesicle RNA cargo analysis throughout human gestation

The contributions of the placenta to normal pregnancy physiology, as well as the pathophysiology of preterm birth, fetal growth disruption, and preeclampsia, are well-recognized, but we cannot predict, prevent, or adequately treat these common complications that affect approximately 10% of human pregnancies. A major challenge to understanding placental development and function using in vivo real-time assessments across gestation (e.g. goal of the Human Placental Project and PAR-22-236) is limited access to longitudinal tissue samples throughout human pregnancy. Others have shown that placental extracellular vesicle (EV) concentrations and contents in maternal plasma may be related to placental development and function, which suggests that placental EVs may be a promising biological resource for in vivo studies. EVs include a range of submicron particles, including small exosomes, larger microvesicles, and cell fragments undergoing necrosis and apoptosis. EVs contain a variety of RNA species (including microRNAs) that provide insights into their source and potential function (e.g. regulating angiogenesis). Methods like density gradient ultracentrifugation, size exclusion chromatography, and affinity capture provide a heterogeneous mixture of EVs from a variety of cell sources and a variety of EV sizes. These methodological shortcomings have limited characterization of placental-specific EVs by failing to precisely characterize their source and cargo. As a result, there is an immediate need for a novel approach to image, count, and isolate cell- and size- specific EVs from maternal blood across gestation to monitor placental development and function. In collaboration with BD Biosciences, we have developed a multiplex high resolution nanoscale flow cytometry method to image, count, isolate and validate placental cell- and size-specific EVs. Importantly, this novel technology enables us to study placental EVs from syncytiotrophoblasts, extravillous trophoblasts, and endovascular trophoblasts that likely have different contents and functions during pregnancy. Moreover, we can compare the content of these sorted EVs with sorted 100nm liposomes spiked into the same plasma to control for background contamination inherent to any EV isolation method. We have also developed a novel 242 monoclonal antibody multiplex design with 3-4 antibodies (3/4-colors)/well in a 96-well plate design to address the entire maternal EV-biome throughout gestation. We hypothesize that nanoFACS sorting technology and our new EV-biome design will bring much needed precision and accuracy to the imaging, counting, and isolation of cell- and size-specific placental EV analysis across gestation, which will help us to better understand the role of EVs in placental development and function in vivo. In this project, we will test for differences in cell- and size-specific EV profiles in maternal plasma, which have already been banked from uncomplicated and complicated pregnancies collected at 12, 24, and 32 weeks’ gestation that are linked to published study subject metrics and unique characterization of uteroplacental blood flow by T2*MRI. Project Number: 1R21HD119452-01 | Fiscal Year: 2025 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Terry Morgan | Institution: OREGON HEALTH & SCIENCE UNIVERSITY, PORTLAND, OR | Award Amount: $429,000 | Activity Code: R21 | Study Section: Pregnancy and Neonatology Study Section[PN] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21HD11945201