Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid

Abstract

Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, nanoparticle tracking analysis, and western blotting. EV uptake and effects were tested in human neonatal enteroids. HMEVs and digesta EVs (dHMEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEVs and dHMEVs contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dHMEVs in part via clathrin-mediated endocytosis. Our data suggest that HMEVs survive to the duodenum and can be absorbed by IECs.