Amniotic Fluid Stem (AFS) cells decrease macroscopic and microscopic intestinal damage, localise to the damaged gut and migrate systemically. (A) Examples of the macroscopic gut appearance of breastfed (BF) rats and necrotising enterocolitis (NEC) rats injected with phosphate buffered saline (PBS) or AFS cells. Gut damage of NEC rats injected with AFS cells (0.4±0.7, n=27) was significantly smaller than in animals injected with PBS (1.4±1.1 n=24, p<0.001), but showed no difference with BF rats (0.1±0.3; n=12, p=n.s.). (B) NEC rats treated with AFS cells revealed significantly less histological damage (0.23±0.8, n=48, p<0.001) in comparison with NEC rats treated with PBS (1.78±0.7, n=50); no damage was observed in BF rats (0.08±0.2, n=12). (C) Macroscopic appearance of bundles of AFS cells injected intraperitoneally on the mesentery and on the gut wall. (D) GFP epifluorescence from AFS cells 24 h after injection was detected in the mesentery and adherent to the serosa of the intestine. At 48 h and 72 h rare AFS cells were found within the villus structure in close junction to the epithelial layer. (E) Sections of rat ileum stained with anti-GFP antibodies to localise AFS cells (green) and anti-smooth muscle actin (SMA) antibodies to label smooth muscle cells (red) (scale bar 20μm). AFS cells coexpressing GFP and SMA were found integrated in the mucosal layer.