Maternal and donor human milk support robust intestinal epithelial growth and differentiation in a fetal intestinal organoid model

Necrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using small intestinal organoids derived from fetal tissue of a gestational age similar to an extremely preterm infant, this study aims to assess the effect of diet on intestinal epithelial growth and differentiation to elucidate the role nutrition type plays in intestinal development and modifies the risk for NEC. Organoids were cultured for 5 days in growth media and 5 days in differentiation media supplemented 1:40 with four different diets: maternal milk (MM), donor human milk (DHM), standard formula, or extensively hydrolyzed formula. Images were captured daily and organoids were quantified. Organoids were preserved for RNA sequencing and immunofluorescence staining with Ki67, cleaved caspase 3, and chromogranin-A. Media was saved for cytokine/chemokine and growth factor analysis.Human milk supplementation improved growth and differentiation of intestinal organoids generating larger organoids during the growth phase and organoids with longer and wider buds during differentiation compared to formula. Ki67 staining confirmed the proliferative nature of milk-supplemented organoids and chromogranin A staining proved that MM-supplemented organoids induced highest enteroendocrine differentiation. Human milk supplementation also upregulated genes involved in proliferation and promoted a homeostatic immune landscape while those supplemented with formula had a downregulation of cell-cycle-promoting genes and a more inflammatory immune signature. Our results show that MM, and to a lesser extent DHM, support robust intestinal epithelial proliferation and differentiation, suggesting a critical role for factors enriched in human milk in intestinal epithelial health.