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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV130057	1/1	Homo sapiens	Placental explant	DG (d)(U)C Filtration	Stenqvist AC	2013	33%
Study summary Full title Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus All authors Stenqvist AC, Nagaeva O, Baranov V, Mincheva-Nilsson L Journal J Immunol Abstract Apoptosis is crucially important in mediating immune privilege of the fetus during pregnancy. We inv (show more...)Apoptosis is crucially important in mediating immune privilege of the fetus during pregnancy. We investigated the expression and in vitro apoptotic activity of two physiologically relevant death messengers, the TNF family members Fas ligand (FasL) and TRAIL in human early and term placentas. Both molecules were intracellularly expressed, confined to the late endosomal compartment of the syncytiotrophoblast, and tightly associated to the generation and secretion of placental exosomes. Using immunoelectron microscopy, we show that FasL and TRAIL are expressed on the limiting membrane of multivesicular bodies where, by membrane invagination, intraluminal microvesicles carrying membranal bioactive FasL and TRAIL are formed and released in the extracellular space as exosomes. Analyzing exosomes secreted from placental explant cultures, to our knowledge, we demonstrate for the first time that FasL and TRAIL are clustered on the exosomal membrane as oligomerized aggregates ready to form death-inducing signaling complex. Consistently, placental FasL- and TRAIL-carrying exosomes triggered apoptosis in Jurkat T cells and activated PBMC in a dose-dependent manner. Limiting the expression of functional FasL and TRAIL to exosomes comprise a dual benefit: 1) storage of exosomal FasL and TRAIL in multivesicular bodies is protected from proteolytic cleavage and 2) upon secretion, delivery of preformed membranal death molecules by exosomes rapidly triggers apoptosis. Our results suggest that bioactive FasL- and TRAIL-carrying exosomes, able to convey apoptosis, are secreted by the placenta and tie up the immunomodulatory and protective role of human placenta to its exosome-secreting ability. (hide) EV-METRIC 33% (62nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Placental explant Sample origin DNF Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C + Filtration Protein markers EV: CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Placental explant Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Density gradient Density medium Sucrose Lowest density fraction 20 Highest density fraction 40 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63 Flow cytometry specific beads Selected surface protein(s) Yes Characterization: Particle analysis Particle analysis: flow cytometry EM EM-type transmission EM/ immune EM Proteïns CD63 Image type Close-up, Wide-field

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