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Collagen IV synthesis is restricted to the enteroendocrine pathway during multilineage differentiation of human colorectal epithelial stem cells

¹ Department of Histopathology, Imperial College School of Medicine, Hammersmith Campus, DuCane Road, London, W12 0NN, UK
² Histopathology Unit, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London, WC2A 3PX, UK

View larger version (108K): [in a new window]   Fig. 1. HRA-19 monolayers grown for 4 days in culture medium containing 10% foetal calf serum-containing (A,C) or serum-free medium (B,D) and stained using immunocytochemistry for either chromogranin A (A,B) or type-IV collagen (C,D). Scattered chromogranin-A-positive endocrine cells are observed only under serum-free conditions (B) in similar numbers to collagen-IV-positive endocrine cells (D), which are also only present in serum-free conditions. Bar, 100 µm.

View larger version (60K): [in a new window]   Fig. 2. Double immunocytochemical staining of an intact monolayer of HRA-19 cells. (A) Chromogranin-A-positive endocrine cells (arrows). (B) Type-IV-collagen staining in the same cells as (A) (arrows). Notice that the staining is restricted to individual cells. Faint staining over the whole monolayer is non-specific background staining, indistinguishable from the no-primary-antibody controls. Bar, 50 µm.

View larger version (110K): [in a new window]   Fig. 3. Immunocytochemical staining for prolyl-4-hydroxylase in an HRA-19 epithelial monolayer grown under serum-free conditions. Positive cells (black) are scattered throughout the monolayer. Bar, 100 µm.

View larger version (14K): [in a new window]   Fig. 4. Time-course analysis of endocrine- and goblet-lineage commitment in HRA-19 cells following the transfer of cells at day 0 to serum-free medium. Total number of cells expressing prolyl-4-hydroxylase and collagen IV in each replicate monolayer can be seen in relation to the numbers of endocrine (chromogranin-A positive) and goblet (colonic-mucus positive) cells.

View larger version (135K): [in a new window]   Fig. 5. Immunocytochemical staining in two adjacent serial sections of normal human colon. Endocrine cells that are PYY positive (A) (arrows) match cells with type-IV-collagen staining in the adjacent section (B) (arrows). Bar, 25 µm.

View larger version (42K): [in a new window]   Fig. 6. Double immunocytochemical staining of whole colonic crypts for chromogranin A and type-IV collagen. Three chromogranin-A-positive endocrine cells are located at the crypt base (arrows) (A). These cells are also shown to be type-IV-collagen positive (arrows) (B). Bar, 25 µm.

View larger version (114K): [in a new window]   Fig. 7. Wax sections of normal human colonic epithelium stained using immunocytochemistry for prolyl-4-hydroxylase. (A) Positive staining is seen in both epithelial cells (wide arrows) and pericryptal mesenchymal cells (thin arrow). (B) Fluorescence microscopy of the same section shows that autofluorescence of endocrine cells colocalises with the epithelial prolyl-4-hydroxylase staining. Bar, 50 µm.

View larger version (123K): [in a new window]   Fig. 8. In situ hybridisation for type-IV collagen on wax sections of normal human colon. Pericryptal fibroblasts, the source of basement-membrane collagen IV, stained positively for collagen mRNA (long arrow). Scattered epithelial cells are also positive for collagen mRNA (arrowheads). Bar, 200 µm.

View larger version (115K): [in a new window]   Fig. 9. A single section of normal human colonic epithelium stained with in situ hybridisation for collagen IV mRNA (A) and immunocytochemistry for chromogranin A (B). A single crypt is shown to contain two collagen-IV mRNA-positive epithelial cells (short arrow) with an associated positively staining pericryptal fibroblast (long arrow) (A). Both collagen-IV mRNA-positive epithelial cells are shown to be chromogranin A positive (B). Bar, 25 µm.

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