Human milk exosomes resist digestion in vitro and are internalized by human intestinal cells

Human milk contains exosomes, i.e. microparticles consisting of microRNAs (miRs) with sizes of ~22 nts. Exosome‐mediated transfer of miRs is a novel mechanism of genetic exchange between cells. miRs bind to the 3′‐untranslated region of target mRNAs and cause translational block or mRNA degradation. We hypothesized that milk exosomes can resist digestion in the gastrointestinal tract and be taken up by enterocytes and thus have the potential to regulate gene expression. Human milk samples from donors at different stages of lactation were collected into tubes with RNAse inhibitor. Exosome RNA isolated by ExoQuick‐TC was eluted in RNAse‐free Exosome lysis buffer. CD9 was used as positive control and calnexin as negative control. miRs of 22 nts extracted from isolated exosomes were verified by BioAnalyzer. Sequencing was done using Illumina HiSeq 2500. miRDeep2 was used to find miRNA signatures from the read alignments to known miR sequences (miRBase) and to quantify expression. Several miRs involved in the immune system, e.g. miR‐146, miR‐181, miR‐21, miR‐16 and let‐7 were found. Only a few miRs were differentially expressed during lactation. In vitro digestion (pepsin + pancreatin) under conditions mimicking those in infants showed a significant proportion of the exosomes surviving digestion as verified by TEM. Human intestinal epithelial cells were incubated with digested exosomes and intracellular localization was determined by anti‐CD9 antibodies using confocal microscopy. The exosomes localized to the nucleus as visualized by Topro3. Thus, human milk exosomes are capable of surviving digestion and being taken up by enterocytes where they localize to the nucleus and may affect gene expression. (Supported by Mead Johnson Nutrition)