@article{cotton_ariel_chen_walcott_dixit_breau_hinesley_kedziora_tang_zheng_et al._2024, title={An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal epithelial cells}, volume={14}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-024-66042-9}, abstractNote={The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Cotton, Michael J. and Ariel, Pablo and Chen, Kaiwen and Walcott, Vanessa A. and Dixit, Michelle and Breau, Keith A. and Hinesley, Caroline M. and Kedziora, Katarzyna M. and Tang, Cynthia Y. and Zheng, Anna and et al.}, year={2024}, month={Jul} }
 @article{ok_liu_bliton_hinesley_pedro_breau_gomez-martinez_burclaff_magness_2023, title={A leaky human colon model reveals uncoupled apical/basal cytotoxicity in early Clostridioides difficile toxin exposure}, volume={324}, ISSN={["1522-1547"]}, DOI={10.1152/ajpgi.00251.2022}, abstractNote={ Novel human colonocyte monolayer cultures, benchmarked by transcriptomics for physiological relevance, detect early cytopathic impacts of Clostridioides difficile toxins TcdA and TcdB. A fluorescent ZO-1 reporter in primary human colonocytes is used to track epithelial barrier disruption in response to TcdA. Basal TcdA/B exposure generally caused more rapid onset and cytotoxicity than apical exposure. Transcriptomics demonstrate changes in tight junction, chemokine, and cytokine receptor gene expression post-TcdA exposure. Diclofenac-induced leaky epithelium enhanced apical exposure toxicity. }, number={4}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY}, author={Ok, Meryem T. and Liu, Jintong and Bliton, R. Jarrett and Hinesley, Caroline M. and Pedro, Ekaterina Ellyce T. San and Breau, Keith A. and Gomez-Martinez, Ismael and Burclaff, Joseph and Magness, Scott T.}, year={2023}, month={Apr}, pages={G262–G280} }
 @article{sarma_catella_san pedro_xiao_durmusoglu_menegatti_crook_magness_hall_2023, title={Design of 8-mer peptides that block <i>Clostridioides difficile</i> toxin A in intestinal cells}, volume={6}, ISSN={["2399-3642"]}, url={https://doi.org/10.1038/s42003-023-05242-x}, DOI={10.1038/s42003-023-05242-x}, abstractNote={AbstractInfections by Clostridioides difficile, a bacterium that targets the large intestine (colon), impact a large number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can be delivered to the gut and inhibit the biocatalytic activity of these toxins represent a promising therapeutic strategy to prevent and treat C. diff. infection. We describe an approach that combines a Peptide Binding Design (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in colon epithelial cells. One peptide, SA1, is found to block TcdA toxicity in primary-derived human colon (large intestinal) epithelial cells. SA1 binds TcdA with a KD of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR).}, number={1}, journal={COMMUNICATIONS BIOLOGY}, author={Sarma, Sudeep and Catella, Carly M. and San Pedro, Ellyce T. and Xiao, Xingqing and Durmusoglu, Deniz and Menegatti, Stefano and Crook, Nathan and Magness, Scott T. and Hall, Carol K.}, year={2023}, month={Aug} }
 @article{rivera_bilton_burclaff_czerwinski_liu_trueblood_hinesley_breau_deal_joshi_et al._2023, title={Hypoxia Primes Human ISCs for Interleukin-Dependent Rescue of Stem Cell Activity}, volume={16}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2023.07.012}, abstractNote={Background and AimsHypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs).MethodshISCs were exposed to <1.0% oxygen in the MPS for 6, 24, 48, and 72 hours. Viability, hypoxia-inducible factor 1a (HIF1a) response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF stabilizers and inhibitors were screened to evaluate HIF-dependent responses.ResultsThe MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72 hours and exhibit peak HIF1a at 24 hours. hISC activity was reduced at 24 hours but recovered at 48 hours. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 IL receptors. Prolyl hydroxylase inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1β, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia.ConclusionsHypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of ILs that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia. Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs). hISCs were exposed to <1.0% oxygen in the MPS for 6, 24, 48, and 72 hours. Viability, hypoxia-inducible factor 1a (HIF1a) response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF stabilizers and inhibitors were screened to evaluate HIF-dependent responses. The MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72 hours and exhibit peak HIF1a at 24 hours. hISC activity was reduced at 24 hours but recovered at 48 hours. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 IL receptors. Prolyl hydroxylase inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1β, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia. Hypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of ILs that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia.}, number={5}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Rivera, Kristina R. and Bilton, R. Jarrett and Burclaff, Joseph and Czerwinski, Michael J. and Liu, Jintong and Trueblood, Jessica M. and Hinesley, Caroline M. and Breau, Keith A. and Deal, Halston E. and Joshi, Shlok and et al.}, year={2023}, pages={823–846} }
 @article{gomez-martinez_bliton_breau_czerwinski_williamson_wen_rawls_magness_2022, title={A Planar Culture Model of Human Absorptive Enterocytes Reveals Metformin Increases Fatty Acid Oxidation and Export}, volume={14}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2022.04.009}, abstractNote={Background & AimsFatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin.MethodsSingle-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (B)–labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography.ResultsSingle-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes.ConclusionsPrimary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role. Fatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin. Single-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (B)–labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography. Single-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes. Primary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.}, number={2}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Gomez-Martinez, Ismael and Bliton, R. Jarrett and Breau, Keith A. and Czerwinski, Michael J. and Williamson, Ian A. and Wen, Jia and Rawls, John F. and Magness, Scott T.}, year={2022}, pages={409–434} }
 @article{min_zhang_bliton_caldwell_caplan_presentation_park_kong_lee_washington_et al._2022, title={Dysplastic Stem Cell Plasticity Functions as a Driving Force for Neoplastic Transformation of Precancerous Gastric Mucosa}, volume={163}, ISSN={["1528-0012"]}, DOI={10.1053/j.gastro.2022.06.021}, abstractNote={BACKGROUND & AIMS
Dysplasia carries a high risk of cancer development, however, the cellular mechanisms for dysplasia evolution to cancer are obscure. We have previously identified two putative dysplastic stem cell (DSC) populations, CD44v6neg/CD133+/CD166+ (DP) and CD44v6+/CD133+/CD166+ (TP), which may contribute to cellular heterogeneity of gastric dysplasia. Here, we investigated functional roles and cell plasticity of non-cancerous Trop2+/CD133+/CD166+ DSCs initially developed in the transition from pre-cancerous metaplasia to dysplasia in the stomach.


METHODS
Dysplastic organoids established from active Kras-induced mouse stomachs were utilized for transcriptome analysis, in vitro differentiation and in vivo tumorigenicity assessments of DSCs. Cell heterogeneity and genetic alterations during clonal evolution of DSCs were examined by next-generation sequencing. Tissue microarrays were used to identify DSCs in human dysplasia. We additionally evaluated the effect of CK1α regulation on the DSC activities using both mouse and human dysplastic organoids.


RESULTS
We identified a high similarity of molecular profiles between DP- and TP-DSCs, but more dynamic activities of DP-DSCs in differentiation and survival for maintaining dysplastic cell lineages through Wnt ligand-independent CK1α/β-catenin signaling. Xenograft studies demonstrated that the DP-DSCs clonally evolve towards multiple types of gastric adenocarcinomas and promote cancer cell heterogeneity by acquiring additional genetic mutations and recruiting the tumor microenvironment. Lastly, growth and survival of both mouse and human dysplastic organoids were controlled by targeting CK1α.


CONCLUSIONS
These findings indicate that the DSCs are de novo gastric cancer-initiating cells responsible for neoplastic transformation and a promising target for intervention in early induction of gastric cancer.}, number={4}, journal={GASTROENTEROLOGY}, author={Min, Jimin and Zhang, Changqing and Bliton, R. Jarrett and Caldwell, Brianna and Caplan, Leah and Presentation, Kimberly S. and Park, Do-Joong and Kong, Seong-Ho and Lee, Hye Seung and Washington, M. Kay and et al.}, year={2022}, month={Oct}, pages={875–890} }
 @article{tse_meganck_araba_yount_shaffer_hou_munt_adams_wykoff_morowitz_et al._2022, title={Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection}, volume={119}, ISSN={["1091-6490"]}, DOI={10.1073/pnas.2118126119}, abstractNote={Significance
          A potential outbreak of swine acute diarrhea syndrome coronavirus (SADS-CoV) in the human population could be devastating. Using genomewide CRISPR knockout screening, we identified the placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection, uncovering a novel antiviral target for CoV infection. The PLAC8-related pathway may also have implications for other CoV infections. Given the ability of SADS-CoV to infect human primary cultures without adaptation, our findings lay the foundation for pandemic preparedness for the potential emergence of SADS-CoVs in response to the One Health Initiative.}, number={18}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Tse, Longping V and Meganck, Rita M. and Araba, Kenza C. and Yount, Boyd L. and Shaffer, Kendall M. and Hou, Yixuan J. and Munt, Jennifer E. and Adams, Lily E. and Wykoff, Jason A. and Morowitz, Jeremy M. and et al.}, year={2022}, month={May} }
 @article{xiao_sarma_menegatti_crook_magness_hall_2021, title={In Silico Identification and Experimental Validation of Peptide-Based Inhibitors Targeting Clostridium difficile Toxin A}, volume={17}, ISSN={["1554-8937"]}, url={https://doi.org/10.1021/acschembio.1c00743}, DOI={10.1021/acschembio.1c00743}, abstractNote={Clostridium difficile infection is mediated by two major exotoxins: toxins A (TcdA) and B (TcdB). Inhibiting the biocatalytic activities of these toxins with targeted peptide-based drugs can reduce the risk of C. difficile infection. In this work, we used a computational strategy that integrates a peptide binding design (PepBD) algorithm and explicit-solvent atomistic molecular dynamics simulation to determine promising toxin A-targeting peptides that can recognize and bind to the catalytic site of the TcdA glucosyltransferase domain (GTD). Our simulation results revealed that two out of three in silico discovered peptides, viz. the neutralizing peptides A (NPA) and B (NPB), exhibit lower binding free energies when bound to the TcdA GTD than the phage-display discovered peptide, viz. the reference peptide (RP). These peptides may serve as potential inhibitors against C. difficile infection. The efficacy of the peptides RP, NPA, and NPB to neutralize the cytopathic effects of TcdA was tested in vitro in human jejunum cells. Both phage-display peptide RP and in silico peptide NPA were found to exhibit strong toxin-neutralizing properties, thereby preventing the TcdA toxicity. However, the in silico peptide NPB demonstrates a relatively low efficacy against TcdA.}, number={1}, journal={ACS CHEMICAL BIOLOGY}, publisher={American Chemical Society (ACS)}, author={Xiao, Xingqing and Sarma, Sudeep and Menegatti, Stefano and Crook, Nathan and Magness, Scott T. and Hall, Carol K.}, year={2021}, month={Dec} }
 @article{arnold_roach_fabela_moorfield_ding_blue_dagher_magness_tamayo_bruno-barcena_et al._2021, title={The pleiotropic effects of prebiotic galacto-oligosaccharides on the aging gut}, volume={9}, ISSN={["2049-2618"]}, DOI={10.1186/s40168-020-00980-0}, abstractNote={AbstractBackgroundPrebiotic galacto-oligosaccharides (GOS) have an extensively demonstrated beneficial impact on intestinal health. In this study, we determined the impact of GOS diets on hallmarks of gut aging: microbiome dysbiosis, inflammation, and intestinal barrier defects (“leaky gut”). We also evaluated if short-term GOS feeding influenced how the aging gut responded to antibiotic challenges in a mouse model ofClostridioides difficileinfection. Finally, we assessed if colonic organoids could reproduce the GOS responder—non-responder phenotypes observed in vivo.ResultsOld animals had a distinct microbiome characterized by increased ratios of non-saccharolytic versus saccharolytic bacteria and, correspondingly, a lower abundance of β-galactosidases compared to young animals. GOS reduced the overall diversity, increased the abundance of specific saccharolytic bacteria (species ofBacteroidesandLactobacillus), increased the abundance of β-galactosidases in young and old animals, and increased the non-saccharolytic organisms; however, a robust, homogeneous bifidogenic effect was not observed. GOS reduced age-associated increased intestinal permeability and increasedMUC2expression and mucus thickness in old mice. Clyndamicin reduced the abundanceBifidobacteriumwhile increasingAkkermansia,Clostridium,Coprococcus,Bacillus,Bacteroides, andRuminococcusin old mice. The antibiotics were more impactful than GOS on modulating serum markers of inflammation. Higher serum levels of IL-17 and IL-6 were observed in control and GOS diets in the antibiotic groups, and within those groups, levels of IL-6 were higher in the GOS groups, regardless of age, and higher in the old compared to young animals in the control diet groups. RTqPCR revealed significantly increased gene expression of TNFα in distal colon tissue of old mice, which was decreased by the GOS diet. Colon transcriptomics analysis of mice fed GOS showed increased expression of genes involved in small-molecule metabolic processes and specifically the respirasome in old animals, which could indicate an increased oxidative metabolism and energetic efficiency. In young mice, GOS induced the expression of binding-related genes. The galectin geneLgals1, a β-galactosyl-binding lectin that bridges molecules by their sugar moieties and is an important modulator of the immune response, and the PI3K-Akt and ECM-receptor interaction pathways were also induced in young mice. Stools from mice exhibiting variable bifidogenic response to GOS injected into colon organoids in the presence of prebiotics reproduced the response and non-response phenotypes observed in vivo suggesting that the composition and functionality of the microbiota are the main contributors to the phenotype.ConclusionsDietary GOS modulated homeostasis of the aging gut by promoting changes in microbiome composition and host gene expression, which was translated into decreased intestinal permeability and increased mucus production. Age was a determining factor on how prebiotics impacted the microbiome and expression of intestinal epithelial cells, especially apparent from the induction of galectin-1 in young but not old mice.}, number={1}, journal={MICROBIOME}, author={Arnold, Jason W. and Roach, Jeffery and Fabela, Salvador and Moorfield, Emily and Ding, Shengli and Blue, Eric and Dagher, Suzanne and Magness, Scott and Tamayo, Rita and Bruno-Barcena, Jose M. and et al.}, year={2021}, month={Jan} }
 @article{arnold_roach_fabela_moorfield_ding_blue_dagher_magness_tamayo_bruno-barcena_et al._2021, title={The pleiotropic effects of prebiotic galacto-oligosaccharides on the aging gut (vol 9, 31, 2021)}, volume={9}, ISSN={["2049-2618"]}, DOI={10.1186/s40168-021-01030-z}, abstractNote={An amendment to this paper has been published and can be accessed via the original article.}, number={1}, journal={MICROBIOME}, author={Arnold, Jason W. and Roach, Jeffery and Fabela, Salvador and Moorfield, Emily and Ding, Shengli and Blue, Eric and Dagher, Suzanne and Magness, Scott and Tamayo, Rita and Bruno-Barcena, Jose M. and et al.}, year={2021}, month={Feb} }
 @article{zwarycz_gracz_rivera_williamson_samsa_starmer_daniele_salter-cid_zhao_magness_2019, title={IL22 Inhibits Epithelial Stem Cell Expansion in an Ileal Organoid Model}, volume={7}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2018.06.008}, abstractNote={Crohn's disease is an inflammatory bowel disease that affects the ileum and is associated with increased cytokines. Although interleukin (IL)6, IL17, IL21, and IL22 are increased in Crohn's disease and are associated with disrupted epithelial regeneration, little is known about their effects on the intestinal stem cells (ISCs) that mediate tissue repair. We hypothesized that ILs may target ISCs and reduce ISC-driven epithelial renewal.A screen of IL6, IL17, IL21, or IL22 was performed on ileal mouse organoids. Computational modeling was used to predict microenvironment cytokine concentrations. Organoid size, survival, proliferation, and differentiation were characterized by morphometrics, quantitative reverse-transcription polymerase chain reaction, and immunostaining on whole organoids or isolated ISCs. ISC function was assayed using serial passaging to single cells followed by organoid quantification. Single-cell RNA sequencing was used to assess Il22ra1 expression patterns in ISCs and transit-amplifying (TA) progenitors. An IL22-transgenic mouse was used to confirm the impact of increased IL22 on proliferative cells in vivo.High IL22 levels caused decreased ileal organoid survival, however, resistant organoids grew larger and showed increased proliferation over controls. Il22ra1 was expressed on only a subset of ISCs and TA progenitors. IL22-treated ISCs did not show appreciable differentiation defects, but ISC biomarker expression and self-renewal-associated pathway activity was reduced and accompanied by an inhibition of ISC expansion. In vivo, chronically increased IL22 levels, similar to predicted microenvironment levels, showed increases in proliferative cells in the TA zone with no increase in ISCs.Increased IL22 limits ISC expansion in favor of increased TA progenitor cell expansion.}, number={1}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Zwarycz, Bailey and Gracz, Adam D. and Rivera, Kristina R. and Williamson, Ian A. and Samsa, Leigh A. and Starmer, Josh and Daniele, Michael A. and Salter-Cid, Luisa and Zhao, Qihong and Magness, Scott T.}, year={2019}, pages={1–17} }
 @article{rivera_pozdin_young_erb_wisniewski_magness_daniele_2019, title={Integrated phosphorescence-based photonic biosensor (iPOB) for monitoring oxygen levels in 3D cell culture systems}, volume={123}, ISSN={["1873-4235"]}, DOI={10.1016/j.bios.2018.07.035}, abstractNote={Physiological processes, such as respiration, circulation, digestion, and many pathologies alter oxygen concentration in the blood and tissue. When designing culture systems to recapitulate the in vivo oxygen environment, it is important to integrate systems for monitoring and controlling oxygen concentration. Herein, we report the design and engineering of a system to remotely monitor and control oxygen concentration inside a device for 3D cell culture. We integrate a photonic oxygen biosensor into the 3D tissue scaffold and regulate oxygen concentration via the control of purging gas flow. The integrated phosphorescence-based oxygen biosensor employs the quenching of palladium-benzoporphyrin by molecular oxygen to transduce the local oxygen concentration in the 3D tissue scaffold. The system is validated by testing the effects of normoxic and hypoxic culture conditions on healthy and tumorigenic breast epithelial cells, MCF-10A cells and BT474 cells, respectively. Under hypoxic conditions, both cell types exhibited upregulation of downstream target genes for the hypoxia marker gene, hypoxia-inducible factor 1α (HIF1A). Lastly, by monitoring the real-time fluctuation of oxygen concentration, we illustrated the formation of hypoxic culture conditions due to limited diffusion of oxygen through 3D tissue scaffolds.}, journal={BIOSENSORS & BIOELECTRONICS}, author={Rivera, Kristina R. and Pozdin, Vladimir A. and Young, Ashlyn T. and Erb, Patrick D. and Wisniewski, Natalie A. and Magness, Scott T. and Daniele, Michael}, year={2019}, month={Jan}, pages={131–140} }
 @article{ferguson_foster_sherry_magness_nielsen_gookin_2019, title={Interferon-λ3 Promotes Epithelial Defense and Barrier Function Against Cryptosporidium parvum Infection}, volume={8}, ISSN={2352-345X}, url={http://dx.doi.org/10.1016/j.jcmgh.2019.02.007}, DOI={10.1016/j.jcmgh.2019.02.007}, abstractNote={Background & AimsThe epithelial response is critical for intestinal defense against Cryptosporidium, but is poorly understood. To uncover the host strategy for defense against Cryptosporidium, we examined the transcriptional response of intestinal epithelial cells (IECs) to C parvum in experimentally infected piglets by microarray. Up-regulated genes were dominated by targets of interferon (IFN) and IFN-λ3 was up-regulated significantly in infected piglet mucosa. Although IFN-λ has been described as a mediator of epithelial defense against viral pathogens, there is limited knowledge of any role against nonviral pathogens. Accordingly, the aim of the study was to determine the significance of IFN-λ3 to epithelial defense and barrier function during C parvum infection.MethodsThe significance of C parvum–induced IFN-λ3 expression was determined using an immunoneutralization approach in neonatal C57BL/6 mice. The ability of the intestinal epithelium to up-regulate IFN-λ2/3 expression in response to C parvum infection and the influence of IFN-λ2/3 on epithelial defense against C parvum invasion, intracellular development, and loss of barrier function was examined using polarized monolayers of a nontransformed porcine-derived small intestinal epithelial cell line (IPEC-J2). Specifically, changes in barrier function were quantified by measurement of transepithelial electrical resistance and transepithelial flux studies.ResultsImmunoneutralization of IFN-λ2/3 in C parvum–infected neonatal mice resulted in a significantly increased parasite burden, fecal shedding, and villus blunting with crypt hyperplasia during peak infection. In vitro, C parvum was sufficient to induce autonomous IFN-λ3 and interferon-stimulated gene 15 expression by IECs. Priming of IECs with recombinant human IFN-λ3 promoted cellular defense against C parvum infection and abrogated C parvum–induced loss of barrier function by decreasing paracellular permeability to sodium.ConclusionsThese studies identify IFN-λ3 as a key epithelial defense mechanism against C parvum infection. The epithelial response is critical for intestinal defense against Cryptosporidium, but is poorly understood. To uncover the host strategy for defense against Cryptosporidium, we examined the transcriptional response of intestinal epithelial cells (IECs) to C parvum in experimentally infected piglets by microarray. Up-regulated genes were dominated by targets of interferon (IFN) and IFN-λ3 was up-regulated significantly in infected piglet mucosa. Although IFN-λ has been described as a mediator of epithelial defense against viral pathogens, there is limited knowledge of any role against nonviral pathogens. Accordingly, the aim of the study was to determine the significance of IFN-λ3 to epithelial defense and barrier function during C parvum infection. The significance of C parvum–induced IFN-λ3 expression was determined using an immunoneutralization approach in neonatal C57BL/6 mice. The ability of the intestinal epithelium to up-regulate IFN-λ2/3 expression in response to C parvum infection and the influence of IFN-λ2/3 on epithelial defense against C parvum invasion, intracellular development, and loss of barrier function was examined using polarized monolayers of a nontransformed porcine-derived small intestinal epithelial cell line (IPEC-J2). Specifically, changes in barrier function were quantified by measurement of transepithelial electrical resistance and transepithelial flux studies. Immunoneutralization of IFN-λ2/3 in C parvum–infected neonatal mice resulted in a significantly increased parasite burden, fecal shedding, and villus blunting with crypt hyperplasia during peak infection. In vitro, C parvum was sufficient to induce autonomous IFN-λ3 and interferon-stimulated gene 15 expression by IECs. Priming of IECs with recombinant human IFN-λ3 promoted cellular defense against C parvum infection and abrogated C parvum–induced loss of barrier function by decreasing paracellular permeability to sodium. These studies identify IFN-λ3 as a key epithelial defense mechanism against C parvum infection.}, number={1}, journal={Cellular and Molecular Gastroenterology and Hepatology}, publisher={Elsevier BV}, author={Ferguson, Sylvia H. and Foster, Derek M. and Sherry, Barbara and Magness, Scott T. and Nielsen, Dahlia M. and Gookin, Jody L.}, year={2019}, pages={1–20} }
 @article{raab_tulasi_wager_morowitz_magness_gracz_2020, title={Quantitative classification of chromatin dynamics reveals regulators of intestinal stem cell differentiation}, volume={147}, ISSN={["1477-9129"]}, DOI={10.1242/dev.181966}, abstractNote={Intestinal stem cell (ISC) plasticity is thought to be regulated by broadly-permissive chromatin shared between ISCs and their progeny. Here, we utilize a Sox9EGFP reporter to examine chromatin across ISC differentiation. We find that open chromatin regions (OCRs) can be defined as broadly-permissive or dynamic in a locus-specific manner, with dynamic OCRs found primarily in loci consistent with distal enhancers. By integrating gene expression with chromatin accessibility at transcription factor (TF) motifs in context of Sox9EGFP populations, we classify broadly-permissive and dynamic chromatin relative to TF usage. These analyses identify known and potential regulators of ISC differentiation via association with dynamic changes in chromatin. Consistent with computational predictions, Id3-null mice exhibit increased numbers of cells expressing ISC-specific biomarker OLFM4. Finally, we examine the relationship between gene expression and 5-hydroxymethylcytosine (5hmC) in Sox9EGFP populations, which reveals 5hmC enrichment in absorptive lineage specific genes. Our data demonstrate that intestinal chromatin dynamics can be quantitatively defined in a locus-specific manner, identify novel potential regulators of ISC differentiation, and provide a chromatin roadmap for further dissecting cis regulation of cell fate in the intestine.}, number={1}, journal={DEVELOPMENT}, author={Raab, Jesse R. and Tulasi, Deepthi Y. and Wager, Kortney E. and Morowitz, Jeremy M. and Magness, Scott T. and Gracz, Adam D.}, year={2020}, month={Jan} }
 @article{gunasekara_speer_wang_nguyen_reed_smiddy_parker_fallon_smith_sims_et al._2018, title={A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies}, volume={90}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.8b02845}, abstractNote={Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as to achieve tighter control of experimental variables than can be achieved with animals. Thus, development of a primary in vitro colonic epithelium cultured as high resistance with transport protein expression and functional behavior similar to that of a native colonic would be of enormous value for pharmaceutical research. A collagen scaffold, in which the degree of collagen cross-linking was present as a gradient, was developed to support the proliferation of primary colonic cells. The gradient of cross-linking created a gradient in stiffness across the scaffold, enabling the scaffold to resist deformation by cells. mRNA expression and quantitative proteomic mass spectrometry of cells growing on these surfaces as a monolayer suggested that the transporters present were similar to those in vivo. Confluent monolayers acted as a barrier to small molecules so that drug transport studies were readily performed. Transport function was evaluated using atenolol (a substrate for passive paracellular transport), propranolol (a substrate for passive transcellular transport), rhodamine 123 (Rh123, a substrate for P-glycoprotein), and riboflavin (a substrate for solute carrier transporters). Atenolol was poorly transported with an apparent permeability ( Papp) of <5 × 10-7 cm s-1, while propranolol demonstrated a Papp of 9.69 × 10-6 cm s-1. Rh123 was transported in a luminal direction ( Papp,efflux/ Papp,influx = 7) and was blocked by verapamil, a known inhibitor of P-glycoprotein. Riboflavin was transported in a basal direction, and saturation of the transporter was observed at high riboflavin concentrations as occurs in vivo. It is anticipated that this platform of primary colonic epithelium will find utility in drug development and physiological studies, since the tissue possesses high integrity and active transporters and metabolism similar to that in vivo.}, number={22}, journal={ANALYTICAL CHEMISTRY}, author={Gunasekara, Dulan B. and Speer, Jennifer and Wang, Yuli and Nguyen, Daniel L. and Reed, Mark I. and Smiddy, Nicole M. and Parker, Joel S. and Fallon, John K. and Smith, Philip C. and Sims, Christopher E. and et al.}, year={2018}, month={Nov}, pages={13331–13340} }
 @misc{wang_kim_hinman_zwarycz_magness_allbritton_2018, title={Bioengineered systems and designer matrices that recapitulate the intestinal stem cell niche}, volume={5}, number={3}, journal={Cellular and Molecular Gastroenterology and Hepatology}, author={Wang, Y. L. and Kim, R. and Hinman, S. S. and Zwarycz, B. and Magness, S. T. and Allbritton, N. L.}, year={2018}, pages={440-} }
 @article{wang_kim_gunasekara_reed_disalvo_nguyen_bultman_sims_magness_allbritton_2018, title={Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer}, volume={5}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2017.10.007}, abstractNote={<h3>Background & Aims</h3> The successful culture of intestinal organoids has greatly enhanced our understanding of intestinal stem cell physiology and enabled the generation of novel intestinal disease models. Although of tremendous value, intestinal organoid culture systems have not yet fully recapitulated the anatomy or physiology of the in vivo intestinal epithelium. The aim of this work was to re-create an intestinal epithelium with a high density of polarized crypts that respond in a physiologic manner to addition of growth factors, metabolites, or cytokines to the basal or luminal tissue surface as occurs in vivo. <h3>Methods</h3> A self-renewing monolayer of human intestinal epithelium was cultured on a collagen scaffold microfabricated with an array of crypt-like invaginations. Placement of chemical factors in either the fluid reservoir below or above the cell-covered scaffolding created a gradient of that chemical across the growing epithelial tissue possessing the in vitro crypt structures. Crypt polarization (size of the stem/proliferative and differentiated cell zones) was assessed in response to gradients of growth factors, cytokines, and bacterial metabolites. <h3>Results</h3> Chemical gradients applied to the shaped human epithelium re-created the stem/proliferative and differentiated cell zones of the in vivo intestine. Short-chain fatty acids applied as a gradient from the luminal side confirmed long-standing hypotheses that butyrate diminished stem/progenitor cell proliferation and promoted differentiation into absorptive colonocytes. A gradient of interferon-γ and tumor necrosis factor-α significantly suppressed the stem/progenitor cell proliferation, altering crypt formation. <h3>Conclusions</h3> The in vitro human colon crypt array accurately mimicked the architecture, luminal accessibility, tissue polarity, cell migration, and cellular responses of in vivo intestinal crypts.}, number={2}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Wang, Yuli and Kim, Raehyun and Gunasekara, Dulan B. and Reed, Mark I. and DiSalvo, Matthew and Nguyen, Daniel L. and Bultman, Scott J. and Sims, Christopher E. and Magness, Scott T. and Allbritton, Nancy L.}, year={2018}, pages={113–130} }
 @article{jin_ibrahim_magness_blikslager_2018, title={Knockout of ClC-2 reveals critical functions of adherens junctions in colonic homeostasis and tumorigenicity}, volume={315}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.00087.2018}, DOI={10.1152/ajpgi.00087.2018}, abstractNote={Adherens junctions (AJs), together with tight junctions (TJs), form an apical junctional complex that regulates intestinal epithelial cell-to-cell adherence and barrier homeostasis. Within the AJ, membrane-bound E-cadherin binds β-catenin, which functions as an essential intracellular signaling molecule. We have previously identified a novel protein in the region of the apical junction complex, chloride channel protein-2 (ClC-2), that we have used to study TJ regulation. In this study, we investigated the possible effects of ClC-2 on the regulation of AJs in intestinal mucosal epithelial homeostasis and tumorigenicity. Mucosal homeostasis and junctional proteins were examined in wild-type (WT) and ClC-2 knockout (KO) mice as well as associated colonoids. Tumorigenicity and AJ-associated signaling were evaluated in a murine colitis-associated tumor model and in a colorectal cancer cell line (HT-29). Colonic tissues from ClC-2 KO mice had altered ultrastructural morphology of intercellular junctions with reduced colonocyte differentiation, whereas jejunal tissues had minimal changes. Colonic crypts from ClC-2 KO mice had significantly higher numbers of less-differentiated forms of colonoids compared with WT. Furthermore, the absence of ClC-2 resulted in redistribution of AJ proteins and increased β-catenin activity. Downregulation of ClC-2 in colorectal cells resulted in significant increases in proliferation associated with disruption of AJs. Colitis-associated tumors in ClC-2 KO mice were significantly increased, associated with β-catenin transcription factor activation. The absence of ClC-2 results in less differentiated colonic crypts and increased tumorigenicity associated with colitis via dysregulation of AJ proteins and activation of β-catenin-associated signaling.NEW & NOTEWORTHY Disruption of adherens junctions in the absence of chloride channel protein-2 revealed critical functions of these junctional structures, including maintenance of colonic homeostasis and differentiation as well as driving tumorigenicity by regulating β-catenin signaling.}, number={6}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={Jin, Younggeon and Ibrahim, Dina and Magness, Scott T. and Blikslager, Anthony T.}, year={2018}, month={Dec}, pages={G966–G979} }
 @article{wang_gunasekara_reed_disalvo_bultman_sims_magness_allbritton_2017, title={A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium}, volume={128}, ISSN={["1878-5905"]}, DOI={10.1016/j.biomaterials.2017.03.005}, abstractNote={The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function.}, journal={BIOMATERIALS}, author={Wang, Yuli and Gunasekara, Duian B. and Reed, Mark I. and DiSalvo, Matthew and Bultman, Scott J. and Sims, Christopher E. and Magness, Scott T. and Allbritton, Nancy L.}, year={2017}, month={Jun}, pages={44–55} }
 @article{bhatt_gunasekara_speer_reed_pena_midkiff_magness_bultman_allbritton_redinbo_2018, title={Nonsteroidal Anti-Inflammatory Drug -Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells}, volume={4}, ISSN={["2373-8227"]}, DOI={10.1021/acsinfecdis.7b00139}, abstractNote={The intestinal epithelium provides a critical barrier that separates the gut microbiota from host tissues. Nonsteroidal anti-inflammatory drugs (NSAIDs) are efficacious analgesics and antipyretics and are among the most frequently used drugs worldwide. In addition to gastric damage, NSAIDs are toxic to the intestinal epithelium, causing erosions, perforations, and longitudinal ulcers in the gut. Here, we use a unique in vitro human primary small intestinal cell monolayer system to pinpoint the intestinal consequences of NSAID treatment. We found that physiologically relevant doses of the NSAID diclofenac (DCF) are cytotoxic because they uncouple mitochondrial oxidative phosphorylation and generate reactive oxygen species. We also find that DCF induces intestinal barrier permeability, facilitating the translocation of compounds from the luminal to the basolateral side of the intestinal epithelium. The results we outline here establish the utility of this novel platform, representative of the human small intestinal epithelium, to understand NSAID toxicity, which can be applied to study multiple aspects of gut barrier function including defense against infectious pathogens and host-microbiota interactions.}, number={1}, journal={ACS INFECTIOUS DISEASES}, author={Bhatt, Aadra P. and Gunasekara, Dulan B. and Speer, Jennifer and Reed, Mark I. and Pena, Alexis N. and Midkiff, Bentley R. and Magness, Scott T. and Bultman, Scott J. and Allbritton, Nancy L. and Redinbo, Matthew R.}, year={2018}, month={Jan}, pages={46–52} }
 @article{wang_disalvo_gunasekara_dutton_proctor_lebhar_williamson_speer_howard_smiddy_et al._2017, title={Self-renewing monolayer of primary colonic or rectal epithelial cells}, volume={4}, number={1}, journal={Cellular and Molecular Gastroenterology and Hepatology}, author={Wang, Y. L. and DiSalvo, M. and Gunasekara, D. B. and Dutton, J. and Proctor, A. and Lebhar, M. S. and Williamson, I. A. and Speer, J. and Howard, R. L. and Smiddy, N. M. and et al.}, year={2017}, pages={165-} }