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n3:pmcid: PMC0
bibo:doi: 10.1016%2Fj.jcmgh.2017.02.011
n9:contains: _:vb71204435 _:vb71204450

Subject Item: _:vb71204435
rdf:type: n9:Section
dc:title: materials and methods
n9:contains: _:vb71204448 _:vb71204449 _:vb71204440 _:vb71204441 _:vb71204442 _:vb71204443 _:vb71204444 _:vb71204445 _:vb71204446 _:vb71204447 _:vb71204436 _:vb71204437 _:vb71204438 _:vb71204439

Subject Item: _:vb71204436
rdf:type: n3:Context
rdf:value: screens. An Lgr5EGFPCreERT2xR26 confetti mouse was used for lineage tracing experiments on the 2-D monolayer. The confetti mouse was injected with 5 mg tamoxifen at 48 hours before death and isolation of crypts from the large intestine.>>18<< Human rectal biopsies were obtained from UNC Hospitals Meadowmont Endoscopy Center with consent of the patient (under the approved UNC Institutional Review Board #14-2013).
n3:mentions: n2:20887898

Subject Item: _:vb71204437
rdf:type: n3:Context
rdf:value: Wnt-3A, R-spondin-2, and noggin were obtained from conditioned media as described previously.>>19<< The concentration of Wnt-3A and R-spondin 2 was measured by using a TCF/LEF luciferase reporter stable HEK293 cell line (Signosis SL-0015; Sunnyvale, CA) with comparison to recombinant Wnt-3A and R-spondin 2 control proteins (R&D Systems
n3:mentions: n2:24113577

Subject Item: _:vb71204438
rdf:type: n3:Context
rdf:value: Isolated crypts were embedded in Matrigel as described previously.>>7<<, 20 Briefly, 10,000 crypts were suspended in 50 μL cold Matrigel (Corning 356235; Corning, NY).
n3:mentions: n2:21889923

Subject Item: _:vb71204439
rdf:type: n3:Context
rdf:value: Isolated crypts were embedded in Matrigel as described previously.7, >>20<< Briefly, 10,000 crypts were suspended in 50 μL cold Matrigel (Corning 356235; Corning, NY).
n3:mentions: n2:24232249

Subject Item: _:vb71204440
rdf:type: n3:Context
rdf:value: (ECM) coatings, tissue culture grade polystyrene was coated with ECM or peptides by incubation at 37°C for 1 hour at a concentration recommended by the vendors. Gelatin (10 mg/mL) was prepared by cross-linking with transglutaminase.>>21<< Polyacrylamide possessed a stiffness of about 800 Pa, and its surface was covalently attached to collagen by sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino) hexanoate as recommended by the manufacturer.
n3:mentions: n2:17641645

Subject Item: _:vb71204441
rdf:type: n3:Context
rdf:value: The 5-ethynyl-2-deoxyuridine (EDU)-based staining was used to reveal proliferative cells.>>22<< Cells were incubated with EDU (10 μmol/L) in medium for 3 hours at 37°C, followed by standard staining (Click-iT EDU Alexa Fluor 647 imaging kit; Thermo Fisher Scientific).
n3:mentions: n2:18272492

Subject Item: _:vb71204442
rdf:type: n3:Context
rdf:value: Primary antibodies were rabbit α-Muc2 (1:200, Santa Cruz sc-15334; Santa Cruz Technology, Santa Cruz, CA),>>23<< rabbit α-ChgA (1:
n3:mentions: n2:21995959

Subject Item: _:vb71204443
rdf:type: n3:Context
rdf:value: Primary antibodies were rabbit α-Muc2 (1:200, Santa Cruz sc-15334; Santa Cruz Technology, Santa Cruz, CA),23 rabbit α-ChgA (1:1000, Abcam ab15160; Cambridge, UK),>>24<< rabbit α-Sox9 (1:
n3:mentions: n2:24647645

Subject Item: _:vb71204444
rdf:type: n3:Context
rdf:value: Primary antibodies were rabbit α-Muc2 (1:200, Santa Cruz sc-15334; Santa Cruz Technology, Santa Cruz, CA),23 rabbit α-ChgA (1:1000, Abcam ab15160; Cambridge, UK),24 rabbit α-Sox9 (1:100, Millipore AB5535; Billerica, MA),>>23<< rabbit α-villin (1:
n3:mentions: n2:21995959

Subject Item: _:vb71204445
rdf:type: n3:Context
rdf:value: Proteintech 20874-1-AP), rabbit α-occludin (1:100, Proteintech 13409-1-AP), rabbit α-NA+/K+-ATPase (1:100, Proteintech 22338-1-AP), rabbit α-integrin-β4 (1:200, Santa Cruz sc-9090), and rabbit α-β-catenin (1:200, Santa Cruz sc-7199).>>25<< The secondary antibodies were donkey anti-rabbit immunoglobulin G conjugated with either Alexa Fluor 488 or 594 (1:
n3:mentions: n2:22388892

Subject Item: _:vb71204446
rdf:type: n3:Context
rdf:value: it is important to assess how much the test compounds and negative controls differ from one another. Strictly standardized mean difference (SSMD) was used to quantify the differences between the dietary compounds and negative controls.>>26<< SSMD is commonly used statistic in compound screens and is calculated as the mean difference between the test sample and reference sample divided by the standard deviation of the difference between the test and reference.
n3:mentions: n2:17517904

Subject Item: _:vb71204447
rdf:type: n3:Context
rdf:value: Sample #78 was used as the negative reference for SSMD calculations for all tissue types. Compound effects were classified by using standard SSMD thresholds>>27<<: |SSMD| ≥ 5 for extremely strong, 5 >|SSMD| ≥ 3 for very strong, 3 >|SSMD| ≥ 2 for strong, 2 > |SSMD| ≥ 1.645 for fairly strong, 1.645 > |SSMD| ≥ 1.28 for moderate, 1.28 > |SSMD| ≥ 1 for fairly moderate, 1 > |SSMD| ≥ 0.75 for fairly weak,
n3:mentions: n2:20397965

Subject Item: _:vb71204448
rdf:type: n3:Context
rdf:value: across all Muc2 screens. Library screens were performed in triplicate across well plates (mouse and Caco-2 monolayers). The SSMD size effect was calculated by using the paired uniformly minimal variance unbiased estimate of SSMD.>>28<< For library screens performed in triplicate within single-well plates (human 2-D monolayers and mouse 3-D organoids), the SSMD size effect was calculated by using the unpaired SSMD under unequal variance.
n3:mentions: n2:21515799

Subject Item: _:vb71204449
rdf:type: n3:Context
rdf:value: 28 For library screens performed in triplicate within single-well plates (human 2-D monolayers and mouse 3-D organoids), the SSMD size effect was calculated by using the unpaired SSMD under unequal variance.>>28<<
n3:mentions: n2:21515799

Subject Item: _:vb71204450
rdf:type: n9:Section
dc:title: results
n9:contains: _:vb71204460 _:vb71204461 _:vb71204462 _:vb71204463 _:vb71204456 _:vb71204457 _:vb71204458 _:vb71204459 _:vb71204452 _:vb71204453 _:vb71204454 _:vb71204455 _:vb71204451 _:vb71204464 _:vb71204465

Subject Item: _:vb71204451
rdf:type: n3:Context
rdf:value: Intestinal stem cells can be maintained as a proliferative culture by providing a medium rich in growth factors and embedding the cells within Matrigel to form 3-D organoids.>>7<< We hypothesized that a 2-D monolayer of epithelial cells would undergo long-term proliferation if provided with the appropriate matrix cues such as a suitable stiffness in addition to the required soluble factors (Figure 1A and B).
n3:mentions: n2:21889923

Subject Item: _:vb71204452
rdf:type: n3:Context
rdf:value: with the appropriate matrix cues such as a suitable stiffness in addition to the required soluble factors (Figure 1A and B). Matrigel is widely used in 3-D stem cell culture systems because it resembles the ECM found in many tissues.>>7<< Crypts cultured on the surface of Matrigel failed to form a spreading 2-D monolayer (Figure 1C).
n3:mentions: n2:21889923

Subject Item: _:vb71204453
rdf:type: n3:Context
rdf:value: ± 0% of crypts, 100 crypts counted per well, n = 3) and possessed morphologies reminiscent of the organoids formed within a Matrigel patty, suggesting that Matrigel did not possess the ECM contacts and stiffness (Young’s modulus ∼ 50 Pa>>29<<) required to allow cells to spread on the surface (Figure 1B).
n3:mentions: n2:21277382

Subject Item: _:vb71204454
rdf:type: n3:Context
rdf:value: Young’s modulus of collagen hydrogel is in the range of 10–1000 Pa,>>30<< depending on source, type, and concentration of collagen and characterization method.
n3:mentions: n2:25284029

Subject Item: _:vb71204455
rdf:type: n3:Context
rdf:value: Intestinal and colonic organoids embedded within Matrigel possess proliferative and differentiated cellular compartments and are widely accepted as an in vitro tissue model for the digestive tract.>>8<< Therefore we sought to compare proliferative capacity and lineage composition between the murine 2-D monolayer and 3-D organoid platforms.
n3:mentions: n2:19329995

Subject Item: _:vb71204456
rdf:type: n3:Context
rdf:value: Mature organoids self-pattern into proliferative and differentiated zones by segregating a high density of stem and progenitor cells to the crypt buds (Figure 3F).>>36<< Although stem cells are self-renewing and persist in the organoid crypt buds, differentiated cells in the organoid have a limited life span and are eventually sloughed into the central pseudolumen.
n3:mentions: n2:20185687

Subject Item: _:vb71204457
rdf:type: n3:Context
rdf:value: these absorptive cells (Figure 5E). At day 3 in culture, all 3-D organoids and 2-D monolayer patches possessed actively proliferating cells (EDU+ and SOX9+), suggesting the presence of stem and progenitor cell lineages (Figure 5G and H).>>23<<, 36, 37 Goblet cells (Muc2+) and enteroendocrine cells (ChgA+) were observed in both human 3-D organoids and 2-D monolayers at day 6 in culture, indicating the presence of differentiated cells (Figure 5G and H).
n3:mentions: n2:21995959

Subject Item: _:vb71204458
rdf:type: n3:Context
rdf:value: absorptive cells (Figure 5E). At day 3 in culture, all 3-D organoids and 2-D monolayer patches possessed actively proliferating cells (EDU+ and SOX9+), suggesting the presence of stem and progenitor cell lineages (Figure 5G and H).23, >>36<<, 37 Goblet cells (Muc2+) and enteroendocrine cells (ChgA+) were observed in both human 3-D organoids and 2-D monolayers at day 6 in culture, indicating the presence of differentiated cells (Figure 5G and H).
n3:mentions: n2:20185687

Subject Item: _:vb71204459
rdf:type: n3:Context
rdf:value: and the control monolayers that lacked exposure to the compounds. Differences between the dietary compounds and negative controls were assessed by using the SSMD, a commonly used statistic to identify potential hits in a library screen.>>26<< SSMD is a measure of the average fold change between a test compound and a negative reference, penalized by the variability of the fold change.
n3:mentions: n2:17517904

Subject Item: _:vb71204460
rdf:type: n3:Context
rdf:value: Figure 1). For example, compounds identified as growth suppressive for the primary cells, eg, many of the fatty acids, nitrates, terpenoids, and curcuminoids, are all known to either promote differentiation or exhibit cell toxicity.>>38<<, 39 Glucoraphanin (#70), responsible for the bitterness of cabbage and Brussels sprouts, reduced epithelial cell numbers to near 0, consistent with its reported toxicity when consumed in large amounts.
n3:mentions: n2:21946166

Subject Item: _:vb71204461
rdf:type: n3:Context
rdf:value: Figure 1). For example, compounds identified as growth suppressive for the primary cells, eg, many of the fatty acids, nitrates, terpenoids, and curcuminoids, are all known to either promote differentiation or exhibit cell toxicity.38, >>39<< Glucoraphanin (#70), responsible for the bitterness of cabbage and Brussels sprouts, reduced epithelial cell numbers to near 0, consistent with its reported toxicity when consumed in large amounts.
n3:mentions: n2:12067580

Subject Item: _:vb71204462
rdf:type: n3:Context
rdf:value: (#75, a microbial metabolite of flaxseed) increased cell numbers relative to that of the control, consistent with its failure to prevent intestinal adenoma formation and pose an increased cancer risk when at very high levels.>>40<< A number of other compounds, such as the phenols gallic acid (#28) and ellagic acid (#29), suppressed the number of cells in S-phase without reducing the number of nuclei.
n3:mentions: n2:16197574

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rdf:value: of cells in S-phase without reducing the number of nuclei. Other molecules such as valproate (#6) and β-carotene (#58) display a similar growth suppressive effect and are recognized in the literature as inducing cell differentiation.>>41<<, 42 Only 9 of 77 compounds (12%) demonstrated increased cell numbers relative to that of the control, with fairly strong or stronger SSMD effect sizes.
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rdf:value: of cells in S-phase without reducing the number of nuclei. Other molecules such as valproate (#6) and β-carotene (#58) display a similar growth suppressive effect and are recognized in the literature as inducing cell differentiation.41, >>42<< Only 9 of 77 compounds (12%) demonstrated increased cell numbers relative to that of the control, with fairly strong or stronger SSMD effect sizes.
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rdf:value: (#42, used in flavorings, fragrances, and cosmetics) and punicalagin (#31, an antioxidant found in pomegranates) pushed murine cells toward the enterocyte lineage reported to block cell proliferation, but by an unknown mechanism.>>43<< Other compounds (phytol, #51, a nuclear-receptor ligand used in the fragrance industry) directed murine cells toward the enterocyte lineage but did not reduce cell proliferation, suggesting a different or multiple modes of action relative
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