Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation

Abstract

We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.

Figure 1

Histological features of human colon tissue in organ culture treated with calcium or multi-mineral extract. Formalin-fixed tissue is hematoxylin- and eosin-stained. Normal colon crypt structure, and stromal integrity, is maintained after 2 d of treatment. Original magnification, ×150.

Figure 2

Immunoperoxidase staining for Ki67 expression in human colon tissue treated with calcium or multi-mineral extract. After 2 d in culture, cell cycling in the crypt is evident by expression of Ki67. Treatments with higher concentrations of calcium or with multi-mineral extract suppress proliferation of crypt cells at sides and base. The figure represents averages ± ranges of two independent experiments. Original magnification, ×150.

Figure 3

Immunoperoxidase staining for E-cadherin expression in human colon tissue treated with calcium or multi-mineral extract. After 2 d in culture, differentiation of the epithelium is assessed by expression of E-cadherin. Treatments with higher concentrations of calcium or with multi-mineral extract induce differentiation further into the crypt—at sides and base. Arrows indicate areas of intense staining (luminal surface, crypt side, and crypt base). The figure represents averages ± ranges of four independent experiments. Original magnification, ×150.

Figure 4

Type I procollagen levels of human colon tissue treated with calcium or multi-mineral extract. Two-day culture supernatants from treated tissue were assayed for procollagen by ELISA. Procollagen levels were dramatically reduced with high calcium or multi-mineral extract treatment. The figure represents averages ± ranges of six independent experiments.

Figure 5

Type I collagen levels of human colon tissue treated with calcium or multi-mineral extract. Two-day culture supernatants from treated tissue were assayed for soluble type I collagen by Western blot analysis. Type I collagen increased with high calcium or multi-mineral extract treatment. The figure represents averages ± ranges of five independent experiments.