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

Abstract

Background and aims 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. Methods 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. Results 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. Conclusions 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.

Differentiation of Human Colon Tissue In Culture: Effects of calcium on trans-epithelial electrical resistance and tissue cohesive properties

“…336 337 Electrical resistance across the polarized gastrointestinal epithelial layer correlates with 338 existence of a barrier to small molecule passage between adjacent epithelial cells (2-4); it is 339 generally accepted that tight junctions, primarily, mediate this property (23). Consistent with 340 occludin expression data -both in the intact colonoids(21) and cells derived from the colonoids 341 (this report) -trans-epithelial electrical resistance was substantial under low-calcium conditions 342…”

“…This is of interest because 347 although several cadherin family members were induced with calcium supplementation, 348 cadherin 1 was not one of them. Rather, cadherin-1 was highly expressed in the low-calcium 349 environment (21,33) and not further induced with calcium supplementation. have also been seen even without overt bowel disease; e.g., as a consequence of high-fat diets 395 (44), hyperglycemia (45), psychological stress (46) and hypoxia (47).…”

“…124 Calcium Chloride was obtained as a 0.5 M solution (PromoCell GmbH, Heidelberg, 125 Germany). Aquamin, a calcium-rich and magnesium-rich multi-mineral product obtained from the 126 skeletal remains of the red marine algae, Lithothamnion sp (22) was provided by Marigot Ltd (Cork,127 Ireland) as a powder and has been used in previous studies (20,21,24,25). Aquamin contains 128 calcium and magnesium in a ratio of approximately 12:1, along with measurable levels of 72 other 129 trace minerals (essentially all of the minerals algae fronds accumulate from the ocean water).…”

“…Adhesion structures, especially desmosomes, 109 were prominent in the treated colonoids. Calcium from either source was comparably effective 110 in stimulating cell-cell adhesion proteins, but Aquamin was more effective than calcium alone at 111 up-regulating basement membrane molecules (21). 112 113 Given the changes in adhesion protein expression, we postulated that improved barrier 114 function and, especially, stronger tissue cohesion would be consequences of intervention.…”

“…In recent studies, we established colonoid cultures from histologically-normal human 92 colon tissue as well as from several large adenomas obtained at endoscopy (20,21). Once 93 established, the colonoid cultures were maintained in a low-calcium (0.15-0.25 mM) 94 environment or exposed to levels of extracellular calcium (1.5 -3.0 mM) that are known to 95 foster epithelial cell differentiation.…”