Research Article: Calcium-induced differentiation in normal human colonoid cultures: Cell-cell / cell-matrix adhesion, barrier formation and tissue integrity

Date Published: April 17, 2019

Publisher: Public Library of Science

Author(s): Durga Attili, Shannon D. McClintock, Areeba H. Rizvi, Shailja Pandya, Humza Rehman, Daniyal M. Nadeem, Aliah Richter, Dafydd Thomas, Michael K. Dame, Danielle Kim Turgeon, James Varani, Muhammad N. Aslam, Gianpaolo Papaccio.


The goal of the study was to assess calcium alone and Aquamin, a multi-mineral natural product that contains magnesium and detectable levels of 72 trace elements in addition to calcium, for capacity to affect growth and differentiation in colonoid cultures derived from histologically-normal human colon tissue.

Colonoid cultures were maintained in a low-calcium (0.25 mM) medium or in medium supplemented with an amount of calcium (1.5–3.0 mM), either from calcium alone or Aquamin for a period of two weeks. This was shown in a previous study to induce differentiation in colonoids derived from large adenomas. Changes in growth, morphological features and protein expression profile were assessed at the end of the incubation period using a combination of phase-contrast and scanning electron microscopy, histology and immunohistology, proteomic assessment and transmission electron microscopy.

Unlike the previously-studied tumor-derived colonoids (which remained un-differentiated in the absence of calcium-supplementation), normal tissue colonoids underwent differentiation as indicated by gross and microscopic appearance, a low proliferative index and high-level expression of cytokeratin 20 in the absence of intervention (i.e., in control condition). Only modest additional changes were seen in these parameters with either calcium alone or Aquamin (providing up to 3.0 mM calcium). In spite of this, proteomic analysis and immunohistochemistry revealed that both interventions induced strong up-regulation of proteins that promote cell-cell and cell-matrix adhesive functions, barrier formation and tissue integrity. Transmission electron microscopy revealed an increase in desmosomes in response to intervention.

These findings demonstrate that colonoids derived from histologically normal human tissue can undergo differentiation in the presence of a low ambient calcium concentration. However, higher calcium levels induce elaboration of proteins that promote cell-cell and cell-matrix adhesion. These changes could lead to improved barrier function and improved colon tissue health.

Partial Text

Epidemiological studies have demonstrated that calcium intake and colon polyp formation / colon cancer are inversely related [1–7]. In spite of this, interventional trials using calcium supplementation to reduce polyp formation have been only modestly effective. Some chemoprevention trials have shown a reduction in polyp incidence [8,9] but others have failed to find significant benefit [10,11]. One chemoprevention study actually found an increased risk of developing colon polyps with the sessile-serrated phenotype—a pathological presentation associated with increased risk of colon cancer—with calcium supplementation [12]. The same interventional trials have also demonstrated only modest (though in some cases, statistically significant) effect on biomarkers of growth and differentiation in histologically-normal colon tissue [13,14].

In the present study, colonoid cultures obtained from histologically-normal colon tissue were examined under low-calcium (0.25 mM) conditions and compared to colonoids treated with a range of calcium concentrations (1.5–3.0 mM) from calcium alone or Aquamin. These interventions were recently shown to induce differentiation and suppress proliferation in adenoma colonoids [33]. Compared to what was observed with adenoma colonoids, where a high proliferation index and minimal differentiation were observed in the absence of intervention (calcium alone or Aquamin), normal tissue colonoids demonstrated a highly-differentiated phenotype in the absence of intervention. Specifically, under both low-calcium conditions and in response to intervention, colonoids appeared either as thin-walled, translucent “cystic” structures or differentiated crypts. The cystic structures reflect colonoids enriched in stem cells and are due to the high level of Wnt-pathway ligands in the culture medium (L-WRN) used to maintain normal tissue colonoids [32–36]. What accounts for the low sensitivity of the adenoma-derived colonoids to low ambient calcium is unclear. While higher calcium (1.5–3.0 mM) from either intervention induced only modest additional differentiation in normal colonoids, adenoma colonoids were highly differentiated in response to these interventions. The potential of epithelial cells in the adenomas to undergo differentiation in response to higher calcium concentrations suggests that even after the premalignant tumors have reached the “large adenoma” size, there is still potential for calcium-supplementation to have a pro-differentiating effect. This is of interest because it has been shown in past studies [54,55] with human colon adenomas that a histologically-differentiated presentation is a favorable prognostic factor.

In summary, the studies described here demonstrate that colonoids obtained from histologically-normal colon tissue express gross and histological features of differentiation in the presence of a low ambient level of extracellular calcium. In this respect, the normal tissue colonoids are different from the previously-studied adenoma colonoids [33], which demonstrated little evidence of differentiation (and a high proliferation index) under low-calcium conditions, but differentiated in response to calcium supplementation. Taken together, these findings suggest that reduced sensitivity to low ambient calcium is an inherent feature of the transformation process in the colonic epithelium. In addition, the present findings show that even in colonoids that are already differentiated based on morphological criteria, intervention with calcium up-regulates proteins that contribute to adhesive function, barrier formation and tissue integrity. To the extent that dietary mineral supplementation can improve barrier formation and tissue integrity in the gastrointestinal tract, it should help reduce chronic inflammation and, ultimately, mitigate some of the age-related conditions that result from chronic inflammation.




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