Neonatal Human Enteroids Take Up Human Milk Extracellular Vesicles That Survive Neonatal Human Digestion
Authors: Yung Claire , Kuhn Madeline , Zhang Yang , Armstrong Randall , Aloia Molly , Olyaei Amy , Scottoline Brian 
Published: May 2024

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. HM contains thousands of bioactive components, including extracellular vesicles (HMEVs). HMEVs are lipid bilayer-encased nanoparticles released from mammary epithelial cells that carry biological cargo to the infant. 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 aims to develop a neonatal enteroid model and EV isolation pipeline to study HMEV-intestinal epithelial cell (IEC) interactions with the ultimate goal of therapy development. Hypothesis: HMEVs survive in vivo neonatal digestion to be taken up by infant IECs. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from post-pyloric tubes. HMEVs were isolated from raw and pasteurized human milk and digesta (n=3 each) 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 (NTA), and western blotting. HMEV sub-populations were examined by super-resolution microscopy. HMEV uptake was tested using apical-out neonatal human enteroids isolated from a 2-month-old patient undergoing surgery for ileal atresia and validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid uptake was assessed with CMTPX dye-labeled-HMEVs, dynamin inhibitor, and confocal microscopy. Results: Our HMEV isolation pipeline reliably isolates HMEVs exhibiting classic EV morphology and enriched in extracellular vesicle markers CD81 and CD9, but depleted of b-casein and lactalbumin from HM and digesta. NTA data indicates that only 17.8 ± 2.9% (p>0.0001) of input HMEVs survive to reach the neonatal intestine. Human mammary gland-derived protein BTN1A1 is present in digesta EVs. Neonatal apical-out human enteroids represent a less proliferative and more differentiated epithelium, e.g. down-regulated KI67 (0.05 ± 0.03-fold relative to basal-out, p<0.0001, n=4-6) and elevated CHGA (14.22 ± 9.30-fold relative to basal-out, p=0.0072, n=4-6) as measured by qRT-PCR and normalized to housekeeping gene RPLP0. Neonatal enteroids take up digested HMEVs largely via dynamin-mediated endocytosis, as measured by CMTPX dye intensity normalized to enteroid area using Fiji (2.05 x 105 ± 2.95 x 104 vs 5.38 x 104 ± 6.08 x 103, p<0.0001, n=62-115 enteroids, HMEV-treatment alone and HMEVs + dynamin inhibitor, respectively).Conclusion: Our data suggest that HMEVs survive to the neonatal intestine and can be absorbed by neonatal enteroids largely via dynamin-mediated endocytosis. These data are an important first step to leveraging HMEVs and their cargo to treat neonatal intestinal inflammation. Funding: NIH K01DK129401, NIH R01HD097367, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.