Cyclic-recombinase-reporter mouse model to determine exosome communication and function during pregnancy

During pregnancy, feto-maternal communication can be mediated through extracellular vesicles, specifically exosomes, 30- to 150-nm 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.

To test exosome trafficking and function, we used a transgenic mouse model containing membrane-targeted, red fluorescent protein tdTomato and enhanced green fluorescent protein cyclic recombinase-reporter construct expressed only in fetal tissues. This model allows fetal tissues and their exosomes to express tdTomato under normal conditions or green fluorescent protein if fetal tissues are exposed to cyclic recombinase that will excise tdTomato. As maternal tissue remains negative for this construct, tdTomato/green fluorescent protein expression and their switching can be used to determine fetal-specific cell and exosome trafficking.

tdTomato/green fluorescent protein-homozygous male mice were mated with wild-type females to have all fetal tissues express the tdTomato/green fluorescent protein allele. Red fluorescence due to tdTomato expression of the tdTomato/green fluorescent protein allele in fetal tissues (placenta, fetal membranes) was confirmed by confocal microscopy on embryonic day 16. Localization of fetal exosomes in maternal uterine tissues were performed by immunostaining for exosome marker CD81 and tdTomato expression followed by confocal microscopy. Fetal exosomes (tdTomato-positive) in maternal plasma were immunoprecipitated using anti-red fluorescent protein tdTomato, followed by confirmation with flow cytometry. To further illustrate the fidelity of fetal exosomes in maternal samples, exosomes bioengineered to contain cyclic recombinase (1.0 × 10¹⁰ exosomes) were injected intraperitoneally on embryonic day 13. On embryonic day 16, fetal (placenta and fetal membranes) tissues were imaged to show tdTomato-to-green fluorescent protein transition. The green fluorescent protein-expressing exomes were localized in maternal tissues (confocal microscopy) and plasma (flow cytometry).

Mating between a male with the tdTomato/green fluorescent protein construct and a null female resulted in fetal tissues and their exosomes expressing tdTomato positivity. Total fetal exosomes in maternal plasma was about 35%. tdTomato-positive exosomes were isolated from maternal plasma and immunostaining localized tdTomato-positive exosomes in maternal uterine tissues. Maternal intraperitoneal injection of cyclic recombinase-enriched exosomes crossed placenta, excised tdTomato from the tdTomato/green fluorescent protein construct in the fetal tissues, and caused green fluorescent protein expression in fetal cells. Furthermore, green fluorescent protein-positive exosomes released from fetal cells were isolated from maternal blood.

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.