Administration of extracellular vesicles derived from human amniotic fluid stem cells: a new treatment for necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease. Amniotic fluid stem cells (AFSC) improve NEC injury but human translation remains difficult. We aimed to evaluate the use of extracellular vesicles (EV) derived from human AFSC. METHODS: Human AFSC (hAFSC) were cultured according to the protocol (Celprogen Inc., California, U.S.A.). Conditioned medium was obtained, ultra-centrifuged, and EV were suspended in phosphate-buffered saline (PBS). C57BL/6 pups were grouped into: (1) breast-fed (Control, n = 11); (2) NEC + placebo (NEC + PBS; n = 10); and (3) NEC + treatment (NEC + EV; n = 11). NEC was induced post-natal days P5-9 by (A) gavage feeding hyperosmolar formula; (B) hypoxia for 10 min; and (C) lipopolysaccharide. Intra-peritoneal injections of PBS or hAFSC-EV were given on P6-7. All animals were sacrificed on P9 and terminal ileum harvested. RESULTS: hAFSC-EV administration reduced intestinal injury (p = 0.0048), NEC incidence (score ≥ 2), and intestinal inflammation (IL-6 p < 0.0001; TNF-α p < 0.0001). Intestinal stem cell expression (Lgr5 +) and cellular proliferation (Ki67) were enhanced above control levels following hAFSC-EV administration (Lgr5 p = 0.0003; Ki67 p < 0.0001). CONCLUSION: hAFSC-EV administration reduced intestinal NEC injury and inflammation while increasing stem cell expression and cellular proliferation. hAFSC-EV administration may induce similar beneficial effects to exogenous stem cells.

Treatment of necrotizing enterocolitis by conditioned medium derived from human amniotic fluid stem cells.

PURPOSE: Necrotizing enterocolitis (NEC) is one of the most distressing gastrointestinal emergencies affecting neonates. Amniotic fluid stem cells (AFSC) improve intestinal injury and survival in experimental NEC but are difficult to administer. In this study, we evaluated whether conditioned medium (CM) derived from human AFSC have protective effects. METHODS: Three groups of C57BL/6 mice were studied: (i) breast-fed mice as control; (ii) experimental NEC mice receiving PBS; and (iii) experimental NEC mice receiving CM. NEC was induced between post-natal days P5 through P9 via: (A) gavage feeding of hyperosmolar formula four-time a day; (B) 10 minutes hypoxia prior to feeds; and (C) lipopolysaccharide administration on P6 and P7. Intra-peritoneal injections of either PBS or CM were given on P6 and P7. All mice were sacrificed on P9 and terminal ileum were harvested for analyses. RESULTS: CM treatment increased survival and reduced intestinal damage, decreased mucosal inflammation (IL-6; TNF-α), neutrophil infiltration (MPO), and apoptosis (CC3), and also restored angiogenesis (VEGF) in the ileum. Additionally, CM treated mice had increased levels of epithelial proliferation (Ki67) and stem cell activity (Olfm4; Lgr5) compared to NEC+PBS mice, showing restored intestinal regeneration and recovery during NEC induction. CM proteomic analysis of CM content identified peptides that regulated immune and stem cell activity. CONCLUSIONS: CM derived from human AFSC administered in experimental NEC exhibited various benefits including reduced intestinal injury and inflammation, increased enterocyte proliferation, and restored intestinal stem cell activity. This study provides the scientific basis for the use of CM derived from AFSC in neonates with NEC.