Figure 3

Expression of intestinal markers in culture. A-D) Continued Cdx2 expression after 7 (A, B) and 11 (C, D) days of culture. E-G) Concentric expression of the columnar epithelial marker cytokeratin 8 (red) and mesenchymal smooth muscle actin (green) persists on day 7 (E, F) and 11 (G) of culture. H-DD) Expression of differentiated epithelial markers. H-M) alkaline phosphatase, visualised by histochemistry, is absent after 1 day of culture (H, I) and is expressed in enterocytes after 7 (J, K) and 11 days (L, M). N-Q) Sucrase-isomaltase, visualised by immunostaining, is absent at 1 (N) and 7 days (O), thereafter demonstrated in enterocytes at day 11 of culture (P, Q). R-T) Goblet cells visualised by PAS staining are seen at 7 days (R, S) and persist for the duration of culture (T). U-X) Chromogranin A, visualised by immunostaining, is expressed in enteroendocrine cells at 7 (U, V) and 11 (W, X) days. Y-AA) The antimicrobial agent lysozyme is demonstrated by immunostaining in Paneth cells at 7 (Y) and 11 (Z, AA) days. BB-DD) HNF-4 α expression, visualised by immunostaining, shows a similar pattern (BB, CC 7 days; DD 11 days of culture). Scale bars = 100 μm A, C, E, H, J, L, N-P, R, T, U, W, Z, BB; 20 μm B, D, F, G, I, K, M, Q, S, V, X, Y, AA, CC, DD.