512 - Establishment of valid necrotizing enterocolitis experimental models and confirmation of MSC-derived EV’s therapeutic efficacy

Poster Number: 512
Publication Number: 512.2111

Background: Necrotizing enterocolitis (NEC) is one of the most critical causes of mortality and morbidity in very low birth weight infants, however, lack of an appropriate therapeutic highly demands its development. We have previously confirmed an enhanced anti-inflammatory, anti-apoptotic, and anti-oxidative function in thrombin-preconditioned Wharton’s-jelly MSC derived extracellular vesicles (EVs).

Objective: This study aimed to establish a valid experimental NEC model that adequately recapitulates the multifactorial and idiopathic pathogenesis of NEC and to investigate the therapeutic efficacy of EVs in NEC model.

Design/Methods: In vitro: Triple-hit Intestinal Epithelial Cell-6 (IEC6) cells (OGD/1%DSS/32℃) were treated with either saline or EVs. LPS and hypoxia were induced to peritoneal macrophages/IEC6 co-culture and treated with either saline or EVs.
In vivo: Postnatal day 4 (P4) ICR mouse pups were orally administered with LPS, gavage fed with hyperosmolar formula and intermittently breast-fed, exposed to hypoxia and hypothermia. At P7, pups were sacrificed for macroscopic, histologic, and biochemical assessment.
Organoid: Intestinal organoid was cultured from P7 mouse small intestine.

Results: Triple-hit IEC6 cells significantly reduced ZO-1 expression compared to CON but was significantly recovered after the EVs treatment, implying an improved gut permeability (p < 0.05). However, epithelial cells alone did not induce inflammation upon triple-hit, which we considered insufficient as an NEC model. We therefore established an immunologically compatible model by co-culturing rat primary peritoneal macrophages with IEC6 cells. LPS/hypoxia-induced co-culture showed a significant increase in inflammatory cytokines IL-1𝛼, IL-1β, IL-6, and TNF-𝛼 compared to CON but were significantly lower with treatment with EVs (p < 0.05), implying EVs’ immunomodulatory effect. In vivo NEC group showed significantly lower body weight, shorter gut length, and higher clinical and histological scores than the CON group (p < 0.01). RT-qPCR showed higher levels of IL-1 β, TNF- 𝛼, IFN-y, LYZ, and MUC2, and lower ZO-1 and OLFM4 levels in NEC group compared to CON group (p < 0.05).

Conclusion(s): In vitro, we confirmed the therapeutic effect of stimulated MSC-derived EVs reducing inflammation in immunologically compatible co-culture model and enhancing gut permeability in triple-hit model. We successfully induced NEC in our in vivo model and established organoids from newborn mice. We are conducting additional injury assessments and confirmation of the therapeutic efficacy of EV using our established models; results are pending.