O1124: Conditioned Medium Derived from Human Amniotic Fluid Stem Cells Promotes Intestinal Regeneration in Experimental Necrotizing Enterocolitis

Background: Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease, commonly observed in premature neonates. NEC is associated with impaired intestinal regeneration and diminished intestinal stem cell (ISC) renewal. Administration of human amniotic fluid stem cell (hAFSC) can re-establish the endogenous stem cell niche during experimental NEC; however, the risks of tumorigenesis as well as engraftment challenge their translation into humans. These challenges can be addressed using conditioned medium derived from human immortalized AFSC (ihAFSC-CM). In this study, we aimed to investigate the regenerative capabilities of ihAFSC-CM in vivo in experimental mouse NEC and ex vivo in mouse intestinal organoids.

Methods: Experimental NEC: ihAFSC-CM was prepared and provided by Micregen Ltd. Neonatal C57BL/6 mice (ethics approval # 44032) from postnatal days (P) 5-9 were divided into three groups: i) healthy control (breast-fed), ii) NEC + phosphate buffered saline (PBS) and iii) NEC + ihAFSC-CM. NEC was induced by gavage feeding of hyperosmolar formula, hypoxia 4 times a day, and lipopolysaccharide (LPS) administration. PBS or ihAFSC-CM were injected intraperitoneally on P6 and P7. Mice were sacrificed on P9, distal ileum was harvested and digested for RT-qPCR.

Organoids: Mouse intestinal organoids were generated from intestinal tissue of healthy P9 C57BL/6 mice. Intestinal crypts were isolated using a gentle dissociation reagent and cultured in Matrigel for 48 hours. To mimic NEC, organoids were then treated with i) LPS + PBS or ii) LPS + ihAFSC-CM and grown in hypoxic conditions for 72 hours. Healthy control organoids were grown at standard conditions.

Results: Administration of ihAFSC-CM in NEC mice significantly increases the mRNA expression of intestinal stem cell markers Lgr5 (p=0.0002) and Olfm4 (p=0.001) compared to NEC + PBS mice. In organoids, the injury led to the formation of more buds protruding from the center of the organoids, indicating increased premature differentiation of stem cells to form the mature cell types within the villi. Administration of ihAFSC-CM resulted in a round organoid morphology, suggesting enhanced stem cell proliferation and reduced differentiation.

Conclusion: Conditioned medium derived from human immortalized AFSC (ihAFSC-CM) restores the stem cell niche in an experimental model of necrotizing enterocolitis. Furthermore, treatment of mouse intestinal organoids with ihAFSC-CM shifts the differentiation-proliferation balance of stem cells towards a proliferative state hence increasing stem cell activity. These findings demonstrate the regenerative capacities of ihAFSC-CM, providing the foundation for future translation as a novel therapeutic strategy for NEC.