Apical-Out Enteroids as an Innovative Model for Necrotizing Enterocolitis

Liebe, Heather; Schlegel, Camille; Cai, Xue; Golubkova, Alena; Loerke, Christopher; Leiva, Tyler; Hunter, Catherine J.

doi:10.1016/j.jss.2022.11.048

Cited by 7 publications

(10 citation statements)

References 26 publications

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“…To test this, we developed a neonatal human enteroid model. Patient‐derived enteroids, enteroid‐derived monolayers, or gut‐on‐a‐chip systems are emerging models for studying neonatal intestinal physiology and pathophysiology (Burge et al., 2022 ; Costello et al., 2022 ; Lanik et al., 2023 ; Liebe et al., 2023 ; Wilson et al., 2022 ). Our model is derived from intestinal epithelial stem cells isolated from a 2‐month‐old participant undergoing surgery for ileal atresia.…”

Section: Resultsmentioning

confidence: 99%

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

Yung,

Zhang,

Kuhn

et al. 2024

J of Extracellular Vesicle

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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 or ostomies. 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, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super‐resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β‐casein and lactalbumin. HMEV and some dEV fractions contain mammary gland‐derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin‐mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.

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Section: Resultsmentioning

confidence: 99%

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

Yung,

Zhang,

Kuhn

et al. 2024

J of Extracellular Vesicle

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show abstract

“…Our neonatal enteroids were propagated in the basal-out orientation where they grow robustly in Matrigel and the basolateral cell surface faces the culture media ( Figure 4a ). For these studies, we used the apical-out enteroid model where the enteroids are cultured in suspension in the absence of a basement membrane (52, 59, 62). This leads to polarity reversal, shown by robust phalloidin staining (green) on the outer surface of the enteroid ( Figure 4a ).…”

Section: Resultsmentioning

confidence: 99%

“…These enteroids grow rapidly and express expected markers of proliferation in the basal-out state. We utilized published protocols (52, 62) to induce polarity reversal in the enteroids allowing direct access to the apical surface from the culture media. We confirmed the apical-out orientation with phalloidin-staining and gene expression showing a significant down-regulation in proliferative markers, consistent with published studies (52).…”

Section: Discussionmentioning

confidence: 99%

“…To test this, we developed a neonatal human enteroid model. Patient-derived enteroids, enteroid-derived monolayers, or gut-on-a-chip systems are emerging models for studying neonatal intestinal physiology and pathophysiology (61)(62)(63)(64)(65). Our model is derived from intestinal epithelial stem cells isolated from a 2-month-old participant undergoing surgery for ileal atresia.…”

Section: Neonatal Human Enteroids Take Up Devsmentioning

confidence: 99%

“…For these studies, we used the apical-out enteroid model where the enteroids are cultured in suspension in the absence of a basement membrane (52,61,64). This leads to polarity reversal, shown by phalloidin staining (green) on the outer surface of the enteroid (Figure 5A).…”

Section: Neonatal Human Enteroids Take Up Devsmentioning

confidence: 99%

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

Yung,

Zhang,

Kuhn

et al. 2023

Preprint

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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.

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Bugs and the barrier: A review of the gut microbiome and intestinal barrier in necrotizing enterocolitis

Snyder

Hunter

2023

Seminars in Pediatric Surgery

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Apical-Out Enteroids as an Innovative Model for Necrotizing Enterocolitis

Cited by 7 publications

References 26 publications

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

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

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

Bugs and the barrier: A review of the gut microbiome and intestinal barrier in necrotizing enterocolitis

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