Body fluid derived exosomes as a novel template for clinical diagnostics

Abstract

Background: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by different cell types from a late endosomal cellular compartment. They can be found in various body fluids including plasma, malignant ascites, urine, amniotic fluid and saliva. Exosomes contain proteins, miRNAs and mRNAs (exosome shuttle RNA, esRNA) that could serve as novel platform for diagnosis.

Method: We isolated exosomes from amniotic fluid, saliva and urine by differential centrifugation on sucrose gradients. Marker proteins were identified by Western blot and FACS analysis after adsorption of exosomes to latex beads. We extracted esRNA from exosomes, carried out RT-PCR, and analyzed amplified products by restriction length polymorphism.

Results: Exosomes were positive for the marker proteins CD24, CD9, Annexin-1 and Hsp70 and displayed the correct buoyant density and orientation of antigens. In sucrose gradients the exosomal fractions contained esRNA that could be isolated with sufficient quantity for further analysis. EsRNAs were protected in exosomes from enzymatic degradation. Amniotic fluid esRNA served as template for the typing of the CD24 single nucleotide polymorphism (rs52812045). It also allowed sex determination of the fetus based on the detection of the male specific ZFY gene product.

Conclusions: Our data demonstrate that exosomes from body fluids carry esRNAs which can be analyzed and offers access to the transcriptome of the host organism. The exosomal lipid bilayer protects the genetic information from degradation. As the isolation of exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostics.

Figure 1

Characterization of amniotic fluid, urine and saliva derived microvesicles. (A) One representative example of microvesicles for amniotic fluid, saliva and urine was analyzed after sucrose density fractionation by Western blot analysis. (B) Isolated exosomes were adsorbed to latex beads and stained for the exosomal markers CD9, CD24 and ADAM10 followed by phycoerythrin-conjugated goat anti-mouse immunoglobulin G and FACS analysis. The negative control represents beads stained with the secondary antibody alone. The gray curve represents the autofluorescence of unstained beads. Note that the staining intensity is proportional to the amount of antigen on the exosomal surface.

Figure 2

Sucrose density analysis of urine derived microvesicles for RNA content. (A) Urinary microvesicles were separated using sucrose density gradient centrifugation. The fractions were harvested, exosomal RNA was isolated and used for cDNA synthesis following RT-PCR analysis. (B) Isolated exosomes were incubated with RNase A alone or in combination with sonication to destroy the exosomal membranes. Isolated esRNA was analyzed by PCR. (C) Total RNA was isolated from amniotic fluid and urine exosomes and analyzed via an Agilent Bioanalyzer. The results show that exosomes contain variable amounts of 18 and 28S rRNAs as well as small and large RNAs.

Figure 3

CD24 SNP analysis using esRNA. (A) Exosomal RNA was used as template for cDNA synthesis following PCR for CD24 and GAPDH. (B) Schematic overview of restriction fragement length polymorphism analysis. (C) CD24 PCR products were digested with BstXI for detection of the CD24 genotype. Note that samples from the same donor have the same number.

Figure 4

Sex determination using amniotic fluid derived esRNA. Twelve different amniotic fluids were analyzed for the gender of the fetus. Exosomal RNA was used as template for cDNA synthesis following gender specific PCR for ZFY. Note that bands running in the front of the gel represent unconsumed primers of the PCR reaction.