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. 2020 Nov 23:8:577221.
doi: 10.3389/fcell.2020.577221. eCollection 2020.

"V体育官网入口" Ulcerative Colitis-Derived Colonoid Culture: A Multi-Mineral-Approach to Improve Barrier Protein Expression

Muhammad N Aslam  1 , Shannon D McClintock  1 , Durga Attili  1 , Shailja Pandya  1 , Humza Rehman  1 , Daniyal M Nadeem  1 , Mohamed Ali H Jawad-Makki  1 , Areeba H Rizvi  1 , Maliha M Berner  2 , Michael K Dame  2 , Danielle Kim Turgeon  2 , James Varani  1
Affiliations

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

Muhammad N Aslam et al. Front Cell Dev Biol. .

Abstract

Background: Recent 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 VSports手机版. The goal of the present study was to determine if the colonic barrier could be improved in tissue from subjects with ulcerative colitis (UC). .

Methods: Colonoid 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. V体育安卓版.

Results: Although 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 V体育ios版. 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. .

Conclusion: A majority of individuals including patients with UC do not reach the recommended daily intake for calcium and other minerals VSports最新版本. 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. .

Keywords: basement membrane; calcium; cell barrier; colonoid; desmosome; organoid culture; proteomics; ulcerative colitis. V体育平台登录.

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Figures

FIGURE 1
FIGURE 1
UC colonoid appearance. Phase-contrast microscopy (A): At the end of the incubation period, intact colonoids were examined by phase-contrast microscopy. Colonoids were present as thick-walled structures with few surface buds. A wide range of sizes and shapes were seen under all conditions. Bar = 200 μm. Scanning electron microscopy (B): Scanning electron microscopy confirmed the presence of smooth surface and few buds in colonoids maintained under low-calcium conditions (Control). Aquamin®-treated colonoids were similar to those maintained in the low-calcium medium. Bar = 100 μm. Histological features (C): At the end of the incubation period, colonoids were examined by light microscopy after staining with hematoxylin and eosin. Under low-calcium conditions (Control), the colonoids were found to be crypts of varying size with a single layer of epithelial cells surrounding a central lumen. Tiny crypts (with as few as 20 cells in cross section) were seen. In the presence of Aquamin®, larger crypts made up of columnar epithelial cells surrounding a large, often irregular-shaped lumen were seen. Goblet cells were apparent. Bar = 100 μm. CK20 expression (D): Immunohistology revealed high-expression of CK20 under all conditions. Bar = 100 μm. Quantification of morphological features and CK20 expression (E,F): Lumen size and wall thickness (E): Means and standard deviations are based on pooled crypts analysis (87–143 individual crypts per condition) of 3 subjects. Asterisks (*) indicate statistical significance from control at p < 0.05. CK20 expression (F): Means and standard deviations are based on n = 3 subjects and 81–120 individual crypts per condition. Asterisks (*) indicate statistical significance from control at p < 0.05. (Subject IDs: Black dots: Subject#1; Green dots: Subject#2; Blue dots: Subject#3).
FIGURE 2
FIGURE 2
Ki67 and cadherin-17 expression by immunohistology. At the end of the incubation period, colonoids were examined after immunostaining of histological sections. Ki67 (A); Bar = 200 μm and Cadherin-17 (B); Bar = 100 μm. Quantitative assessment of Ki67 staining (C) is based on nuclear algorithm (v9) and pooled data represent means and standard deviations from n = 3 subjects and 36–78 individual crypts per condition. Asterisks (*) indicate statistical significance from control at p < 0.05. Cadherin-17 (D) values represent positivity (measured using Positive Pixel Value v9). Means and standard deviations are based on n = 3 subjects and 68–124 individual crypts per condition. Asterisks (*) indicate statistical significance from control at p < 0.05. CDH17: Cadherin-17. (Subject IDs: Black dots: Subject#1; Green dots: Subject#2; Blue dots: Subject#3).
FIGURE 3
FIGURE 3
Desmoglein-2 and desmosomes. Immunohistology (A): At the end of the incubation period, tissue sections were stained for desmoglein-2 and examined. Staining was diffuse and intracellular in colonoids maintained under low-calcium conditions. Staining was more intense in sections from Aquamin®-treated colonoids. Staining was prominent along the basolateral border in treated colonoids as seen in the inset. Bar = 200 μm, inset bar = 50 μm. Transmission electron microscopy (B): At the end of the incubation period, ultra-thin sections were examined for desmosomes and other cell surface structures. Desmosomes were present in all conditions (white arrows) but a higher density of desmosomes along the lateral surface (cellular junctions between two cells) could be seen with intervention. Under all conditions, tight junctions were evident on the luminal surface (black arrows and insets). Magnification: 5,000X; Bars = 600 nm. Quantification of desmoglein-2 expression (C) and desmosome counts (D): Immunostaining (C) results are means and standard deviations based on pooled crypt data from 3 subjects and 85–139 individual crypts per condition. Asterisks (*) indicate statistical significance from control at p < 0.05. Quantitative TEM (D). The desmosome count was conducted at 5000X (n = 3 subjects with 7–18 ultra-structural images per subject) to obtain the actual number (means and SD) of desmosomes present in each high-power section. Asterisks indicate statistical significance from control at p < 0.05 level. (Subject IDs: Black dots: Subject#1; Green dots: Subject#2; Blue dots: Subject#3)
FIGURE 4
FIGURE 4
Quantitative proteomics analysis of ulcerative colitis tissue derived colonoids. (A) Heatmap of the 308 differentially expressed proteins that are significantly significant (p < 0.05) across all subjects (n = 3) and all culture conditions. (A) Complete list of these proteins are presented in Supplementary Table 5. Significantly enriched GO molecular functions (B) and GO biological processes (C) involving these proteins are shown. STRING-database (v11) was used for these enrichment analyses. For these graphs, observed genes (bars) and percentage of these genes as compared to the all involved genes (blue line) are plotted on the left y-axis. While false discovery rate is plotted on the right y-axis (green line). The GO annotation – molecular functions (78 functions) and biological processes (230 processes) are placed on the x-axis and listed in Supplementary Table 6.
FIGURE 5
FIGURE 5
Schematic representation of the colonic mucosa in UC-derived colonoid culture and structural changes due to intervention with Aquamin®. Tight junctions are observed at the apical surface between adjacent cells in both control and treated colonoids; there is little observable difference between the two. Desmosomes (shown along the lateral surface between cells) are increased in response to treatment. This should support increased tissue strength. Additional changes resulting from Aquamin® intervention include an increase in the non-collagenous components of the basement membrane and an increase in hemidesmosomal proteins. These changes should promote improved cell-matrix adhesion. Increased mucin and trefoil levels, leading to a thicker mucous layer at the luminal surface, should contribute to more efficient trapping of bacteria. In aggregate, these changes should provide for improved barrier function and may help mitigate colonic inflammation.
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