Cre-Reporter Mouse Model to Determine Exosome Communication and Function during Pregnancy

Abstract Background During pregnancy, feto-maternal communication can be mediated through extracellular vesicles, specifically exosomes, 30-150 nm size particles released from each cell. Exosomes carry cellular signals and traffic between fetal and maternal tissues to produce functional changes in recipient cells. Exosomes may function as a biomarker indicative of the physiologic status of their tissue of origin. These properties of exosomes during pregnancy are not well studied. Objective To test exosome trafficking and function, we utilized a transgenic mouse model containing membrane-targeted, red fluorescent protein tdTomato (mT) and enhanced green fluorescent protein (mG) Cyclic recombinase (Cre)-reporter construct expressed only in fetal tissues. This model allows fetal tissues and their exosomes to express mT under normal conditions or mG expression if fetal tissues are exposed to Cre that will excise mT. As maternal tissue remains negative for this construct, mT/mG expression and their switching can be utilized to determine fetal specific cell and exosome trafficking. Study Design mT/mG homozygous males were mated with wild type (WT) females to have all fetal tissues express the mT/mG allele. Red florescence due to mT expression of the mT/mG allele in fetal tissues (placenta, fetal membranes) was confirmed by confocal microscopy on embryonic day (E) 16. Localization of fetal exosomes in maternal uterine tissues were performed by immunostaining for exosome marker CD81 and mT expression followed by confocal microscopy. Fetal exosomes (mT+) in maternal plasma were immunoprecipitated using anti-mT and followed by confirmation by flow cytometry. To further illustrate the fidelity of fetal exosomes in maternal samples, exosomes bioengineered to contain Cre (1.0x10 10 exosomes) were injected intraperitoneally (IP) on E13. On E16, fetal (placenta and fetal membranes) tissues were imaged to show mT to mG transition.mG expressing exomes were localized in maternal tissues (confocal microscopy) and plasma (flow cytometry). Results Mating between male with the mT/mG construct and null female resulted in fetal tissues and their exosomes expressing mT+. Total fetal exosomes in maternal plasma was about 35%. mT+ exosomes were isolated from maternal plasma and immunostaining localized mT+ exosomes in maternal uterine tissues. Maternal IP injection of Cre-enriched exosomes crossed placenta, excised mT from the mT/mG construct in the fetal tissues and caused mG expression in fetal cells. Further, mG+ exosomes released from fetal cells were isolated from maternal blood. Conclusion In this pilot study, we report feto-maternal and maternal-fetal trafficking of exosomes indicative of paracrine signaling during pregnancy. Exosomes from the maternal side can produce functional changes in fetal tissues. Trafficking of exosomes suggests their potential role in pregnancy as biomarkers of fetal functions and usefulness as a carrier of drugs and other cargo to the fetal side during pregnancy. Isolation and characterization of fetal exosomes can advance fetal research without performing invasive procedures.