Division of Maternal-Fetal Medicine & Perinatal Research, Department of Obstetrics & Gynecology, The University of Texas Medical Branch, Galveston, TX.
ILIAS Biologics, Inc., Daejeon, Republic of Korea.
Am J Obstet Gynecol. 2019 Nov;221(5):502.e1-502.e12. doi: 10.1016/j.ajog.2019.06.010. Epub 2019 Jun 14.
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 × 10 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.
在怀孕期间，胎儿-母体的通讯可以通过细胞外囊泡（特别是外泌体）介导，外泌体是一种 30-150nm 的颗粒，由每个细胞释放。外泌体携带细胞信号，并在胎儿和母体组织之间运输，从而在受体细胞中产生功能变化。外泌体可以作为其来源组织生理状态的生物标志物。这些外泌体在怀孕期间的特性尚未得到很好的研究。
为了测试外泌体的运输和功能，我们使用了一种转基因小鼠模型，该模型含有膜靶向的红色荧光蛋白 tdTomato 和增强型绿色荧光蛋白环状重组酶报告构建体，仅在胎儿组织中表达。该模型允许胎儿组织及其外泌体在正常条件下表达 tdTomato，或者如果胎儿组织暴露于环状重组酶，则表达绿色荧光蛋白，该酶将切除 tdTomato。由于母体组织中不存在该构建体，因此可以使用 tdTomato/绿色荧光蛋白的表达及其转换来确定胎儿特异性细胞和外泌体的运输。
将 tdTomato/绿色荧光蛋白纯合子雄性小鼠与野生型雌性小鼠交配，使所有胎儿组织表达 tdTomato/绿色荧光蛋白等位基因。通过在胚胎第 16 天进行共聚焦显微镜检查，确认 tdTomato/green fluorescent protein 等位基因在胎儿组织（胎盘、胎膜）中的红色荧光 tdTomato 表达。通过免疫染色外泌体标记物 CD81 和 tdTomato 表达，然后进行共聚焦显微镜检查，检测胎儿外泌体在母体子宫组织中的定位。使用抗红色荧光蛋白 tdTomato 对母体血浆中的胎儿外泌体（tdTomato 阳性）进行免疫沉淀，然后通过流式细胞术进行确认。为了进一步说明母体样本中外泌体的保真度，我们将含有环状重组酶的外泌体（1.0×10 个外泌体）生物工程化后注入胚胎第 13 天的腹膜内。在胚胎第 16 天，对胎儿（胎盘和胎膜）组织进行成像，以显示 tdTomato 到绿色荧光蛋白的转变。在母体组织（共聚焦显微镜）和血浆（流式细胞术）中定位表达绿色荧光蛋白的外泌体。
tdTomato/green fluorescent protein 构建体的雄性与 null 雌性交配，导致胎儿组织及其外泌体表达 tdTomato 阳性。母体血浆中的总胎儿外泌体约为 35%。从母体血浆中分离出 tdTomato 阳性的外泌体，并对其进行免疫染色，将 tdTomato 阳性的外泌体定位在母体子宫组织中。母体腹腔注射富含环状重组酶的外泌体后，穿过胎盘，从胎儿组织中的 tdTomato/green fluorescent protein 构建体中切除 tdTomato，并在胎儿细胞中引起绿色荧光蛋白表达。此外，从母体血液中分离出从胎儿细胞释放的绿色荧光蛋白阳性的外泌体。
在这项初步研究中，我们报告了指示妊娠期间旁分泌信号的胎儿-母体和母体-胎儿外泌体的运输。来自母体的外泌体可以对胎儿组织产生功能变化。外泌体的运输表明它们在外周血中作为胎儿功能的生物标志物和作为药物和其他货物载体向胎儿侧的潜在作用。胎儿外泌体的分离和表征可以推进胎儿研究，而无需进行侵入性程序。
