Ulcerative Colitis-Derived Colonoid Culture: A Multi-Mineral-Approach to Improve Barrier Protein Expression

Abstract

BackgroundRecent studies demonstrated that Aquamin®, a calcium-, magnesium-rich, multi-mineral natural product, improves barrier structure and function in colonoids obtained from the tissue of healthy subjects. The goal of the present study was to determine if the colonic barrier could be improved in tissue from subjects with ulcerative colitis (UC).MethodsColonoid cultures were established with colon biopsies from 9 individuals with UC. The colonoids were then incubated for a 2-week period under control conditions (in culture medium with a final calcium concentration of 0.25 mM) or in the same medium supplemented with Aquamin® to provide 1.5 – 4.5 mM calcium. Effects on differentiation and barrier protein expression were determined using several approaches: phase-contrast and scanning electron microscopy, quantitative histology and immunohistology, mass spectrometry-based proteome assessment and transmission electron microscopy.ResultsAlthough there were no gross changes in colonoid appearance, there was an increase in lumen diameter and wall thickness on histology and greater expression of cytokeratin 20 (CK20) along with reduced expression of Ki67 by quantitative immunohistology observed with intervention. In parallel, upregulation of several differentiation-related proteins was seen in a proteomic screen with the intervention. Aquamin®-treated colonoids demonstrated a modest up-regulation of tight junctional proteins but stronger induction of adherens junction and desmosomal proteins. Increased desmosomes were seen at the ultrastructural level. Proteomic analysis demonstrated increased expression of several basement membrane proteins and hemidesmosomal components. Proteins expressed at the apical surface (mucins and trefoils) were also increased as were several additional proteins with anti-microbial activity or that modulate inflammation. Finally, several transporter proteins that affect electrolyte balance (and, thereby affect water resorption) were increased. At the same time, growth and cell cycle regulatory proteins (Ki67, nucleophosmin, and stathmin) were significantly down-regulated. Laminin interactions, matrix formation and extracellular matrix organization were the top three up-regulated pathways with the intervention.ConclusionA majority of individuals including patients with UC do not reach the recommended daily intake for calcium and other minerals. To the extent that such deficiencies might contribute to the weakening of the colonic barrier, the findings employing UC tissue-derived colonoids here suggest that adequate mineral intake might improve the colonic barrier.

A MULTI-MINERAL INTERVENTION TO MODULATE COLONIC MUCOSAL PROTEIN PROFILE: Results from a 90-day trial in healthy human subjects

“…Due to these limitations, several (though not all) of the potentially important outcomes demonstrated beneficial trends but failed to reach a statistically significant level. Partially off-setting this limitation was the striking correlation between findings presented here and results from earlier studies in the colon organoid model [22][23][24].…”

“…Although the major focus of this effort was colon polyp prevention, our findings allow us to suggest that benefits of multi-mineral intervention may extend beyond this indication. Specifically, the up-regulation of multiple adhesion molecules, both in the interventional model described here and in organoid culture model [22][23][24][25], may translate into improved colon mucosal barrier. In the organoid culture model, it should be noted, exposure to Aquamin ® resulted in increased trans-epithelial electrical resistance and increased tissue cohesion in parallel with adhesion molecule and barrier protein upregulation [25].…”

“…While our past preclinical studies [20][21][22][23][24][25][26][27] have provided compelling evidence that a multi-mineral All rights reserved. No reuse allowed without permission.…”

“…Aquamin ® is derived from the mineralized remains of red marine algae and consists of calcium and magnesium along with detectable levels of 72 additional trace elements [20][21][22][23][24][25][26][27]. In regard to colon cancer, specifically, we have shown that Aquamin ® reduces proliferation and increases differentiation of human colon carcinoma cells in monolayer culture [20,21] and in organoid culture [22][23][24][25] more effectively than calcium alone. Along with these in vitro studies, two long-term (15-18 month) studies in mice demonstrated the improved efficacy of Aquamin ® relative to that of calcium alone for inhibition of colon polyp formation [26,27].…”

“…Findings from the interventional trial are compared to recent findings obtained using similar approaches in colon organoid culture [22][23][24].…”