Amniotic Fluid Stem (AFS) cells lengthen survival and decrease morbidity in rats with Necrotising enterocolitis (NEC) by preserving gut function. (A) Experimental design. (B) Bone marrow mesenchymal stem cells (BM-MSCs)-treated NEC rats had a similar survival rate at 7 days of life as control NEC rats injected with phosphate buffered saline (PBS) (p=ns), while breastfed (BF) rats survived significantly longer than both groups (p<0.0001). AFS cells-treated NEC rats (n=40) had a significantly higher survival rate at 7 days of life than NEC rats treated with BM-MSCs (n=17; p=0.024), PBS (n=24; p<0.0001) or myoblasts (p<0.0001). (C) This effect of AFS cells was extremely reproducible, as cumulative results of several experiments showed a consistent survival benefit (AFS cells n=121 vs PBS n=120, p<0.0001). (D) Morbidity analysis, evaluated using a validated clinical sickness score, confirmed a significant benefit of AFS cell treatment in comparison with PBS (AFS cells 2.0±1.6 vs PBS 3.7±2.1, p<0.01), although AFS cell-treated NEC rats showed a worse outcome compared with BF rats (BF 0.2±0.39, p<0.01 vs AFS cell rats, p<0.001 vs PBS rats). (E) MRI of AFS cells-treated NEC rats (left column of images) and untreated rats (right column of images). Row 1 (i. and ii.): degree of ascites measured using T2 maps: the total number of voxels with T2>160 ms identified as dark red regions which indicates areas of fluid accumulation were different between the PBS (1682±453) and AFS (224±135, p<0.05) groups which did not differ from the BF (278±27). Row 2 (iii. and iv.): bowel wall thickness using µMRI images: marked structural changes were observed in the untreated rats. Row 3 (v. and vi.): representative axial slices demonstrate a similar pattern. Row 4 (vii. and viii.): magnified images of bowel loops from the respective axial slices highlight the loss of bowel wall integrity in the untreated rats. Row 5 (ix. and x.): representative axial slices of BF rats and magnified image of bowel loop showing normal intestinal architecture. (F) Carmine red solution administration revealed that motility was decreased in NEC rats injected with PBS (p<0.001) but it was normal in rats injected with AFS cells (p=n.s.) when compared with BF. (G) Intestinal permeability, measured as plasma lactulose/mannitol ratio: in comparison with BF rats (0.004±0.002, n=9), PBS rats also had a significant increase in intestinal permeability (0.043±0.004; n=21, p<0.001) which was restored in AFS rats (0.031±0.004; n=25, p<0.05).