Fig 1: Isolation, characterization, and differentiation of swine ENSCs. (A) Small intestinal muscularis propria was isolated by microdissection. (B) Cross-sections show PGP9.5+ myenteric neurons between the SMA-expressing circular muscle (CM) and longitudinal muscle (LM). Following tissue digestion and culture for 10 days, enteric neurospheres are formed (C). Immunohistochemical characterization confirms presence of neurons expressing Hu (D, D’), smooth muscle (E); and glial cells expressing P75 (F), NCAM (G), GFAP (H), Tuj1 (I), and Sox10 (J, J’). After neurosphere dissociation, cells were cultured on a fibronectin-coated surface, where Tuj1+ neurons (K, L) and P75+/S100B+ (K, M) glia are seen. Scale bars: A, 1 cm; C, 100 µm; L, 50 µm. ENSC: enteric neuronal stem cell; GFP: green fluorescent protein; SMA: smooth muscle actin.