It is useful to be able to grow enriched populations of stem cells in vitro. Growth of stem cells as tissue spheroids is a key methodology permitting sustainable culture of adult epithelial cells. ...Gastrointestinal stem cells can be propagated by using conditioned medium from a supportive cell line (L-WRN). This protocol describes how to prepare conditioned medium and how to culture stem cell-enriched epithelial spheroids from the mouse gastrointestine. These spheroids are also amenable to genetic modification with recombinant lentiviruses. This system enables many types of cell biological assays that have been performed with immortalized cell lines to be applied to spheroids. Isolation of epithelial cell units from mice takes up to 2 h, and stem cell-enriched gastrointestinal spheroids are obtained within 3 d. Genetically modified spheroids with lentiviruses can be obtained in 2 weeks.
Mammals are defined by their metagenome, a combination of host and microbiome genes. This knowledge presents opportunities to further basic biology with translation to human diseases. However, the ...now-documented influence of the metagenome on experimental results and the reproducibility of in vivo mammalian models present new challenges. Here we provide the scientific basis for calling on all investigators, editors and funding agencies to embrace changes that will enhance reproducible and interpretable experiments by accounting for metagenomic effects. Implementation of new reporting and experimental design principles will improve experimental work, speed discovery and translation, and properly use substantial investments in biomedical research.
We are currently in an exciting time when our understanding of genetic underpinnings of inflammatory bowel disease (IBD) has undergone a revolution, based in large part on novel genotyping and ...sequencing technologies. With >160 susceptible loci identified for IBD, the goal is now to understand at a fundamental level the function of these susceptibility alleles. Determining the clinical relevance of how these susceptible genes shape the development of IBD is also a high priority. The main challenge is to understand how the environment and microbiome play a role in triggering disease in genetically susceptible individuals, as the interactions may be complex. To advance the field, novel in vitro and mouse models that are designed to interrogate complex genetics and functionally test hypotheses are needed. Ultimately, the goal of genetics studies will be to translate genetics to patients with IBD and improve their care.
The process of wound repair in epithelium-lined organs of mammals is complex and is influenced by numerous secreted factors including cytokines, growth factors, and chemokines. However, the cellular ...organizers of this process are still not understood. Recent studies of tissue regeneration in organisms with simpler development have uncovered details about the activity of stem cells in the mesenchyme (the blastema) during this process. These blastemal cells are well positioned to interpret cues from the environment and to execute decisions about the direction of wound repair. In mammalian wounds, stromal stem cells appear to be positioned to perform functions similar to those of blastemal cells, including communication with both the overlying epithelium and the inflammatory cells in the mesenchyme.
Despite the accepted health benefits of consuming dietary fiber, little is known about the mechanisms by which fiber deprivation impacts the gut microbiota and alters disease risk. Using a ...gnotobiotic mouse model, in which animals were colonized with a synthetic human gut microbiota composed of fully sequenced commensal bacteria, we elucidated the functional interactions between dietary fiber, the gut microbiota, and the colonic mucus barrier, which serves as a primary defense against enteric pathogens. We show that during chronic or intermittent dietary fiber deficiency, the gut microbiota resorts to host-secreted mucus glycoproteins as a nutrient source, leading to erosion of the colonic mucus barrier. Dietary fiber deprivation, together with a fiber-deprived, mucus-eroding microbiota, promotes greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. Our work reveals intricate pathways linking diet, the gut microbiome, and intestinal barrier dysfunction, which could be exploited to improve health using dietary therapeutics.
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•Characterized synthetic bacterial communities enable functional insights in vivo•Low-fiber diet promotes expansion and activity of colonic mucus-degrading bacteria•Purified prebiotic fibers do not alleviate degradation of the mucus layer•Fiber-deprived gut microbiota promotes aggressive colitis by an enteric pathogen
Regular consumption of dietary fiber helps prevent erosion of the intestinal mucus barrier by the gut microbiome, blunting pathogen infection and reducing the incidence of colitis.
In the mammalian intestine, crypts of Leiberkühn house intestinal epithelial stem/progenitor cells at their base. The mammalian intestine also harbors a diverse array of microbial metabolite ...compounds that potentially modulate stem/progenitor cell activity. Unbiased screening identified butyrate, a prominent bacterial metabolite, as a potent inhibitor of intestinal stem/progenitor proliferation at physiologic concentrations. During homeostasis, differentiated colonocytes metabolized butyrate likely preventing it from reaching proliferating epithelial stem/progenitor cells within the crypt. Exposure of stem/progenitor cells in vivo to butyrate through either mucosal injury or application to a naturally crypt-less host organism led to inhibition of proliferation and delayed wound repair. The mechanism of butyrate action depended on the transcription factor Foxo3. Our findings indicate that mammalian crypt architecture protects stem/progenitor cell proliferation in part through a metabolic barrier formed by differentiated colonocytes that consume butyrate and stimulate future studies on the interplay of host anatomy and microbiome metabolism.
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•Microbial metabolite screening identifies intestinal stem cell effectors•Butyrate suppresses intestinal stem cell proliferation upon exposure•Crypt structure and colonocytes protect stem/progenitor cells
The architecture of intestinal crypts protects the stem cells at their base from a growth-inhibiting metabolite derived from the gut microbiome. Might these findings suggest co-evolution of mammalian anatomy with commensal flora?
IL-6 is a pleiotropic cytokine often associated with inflammation. Inhibition of this pathway has led to successful treatment of rheumatoid arthritis, but one unforeseen potential complication of ...anti-IL-6 therapy is bowel perforation. Within the intestine, IL-6 has been shown to prevent epithelial apoptosis during prolonged inflammation. The role of IL-6 in the intestine during an initial inflammatory insult is unknown. Here, we evaluate the role of IL-6 at the onset of an inflammatory injury. Using two murine models of bowel injury - wound by biopsy and bacterial triggered colitis - we demonstrated that IL-6 is induced soon after injury by multiple cell types including intraepithelial lymphocytes. Inhibition of IL-6 resulted in impaired wound healing due to decreased epithelial proliferation. Using intestinal tissue obtained from patients who underwent surgical resection of the colon due to traumatic perforation, we observed cells with detectable IL-6 within the area of perforation and not at distant sites. Our data demonstrate the important role of IL-6 produced in part by intraepithelial lymphocytes at the onset of an inflammatory injury for epithelial proliferation and wound repair.
The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualised medicine and ...research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (ie, barrier function and host-microbial interactions).
We created a large panel of human gastrointestinal epithelial cell lines (n=65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays.
We obtained epithelial lines from all accessible tissue sites within 2 weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3-1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarised monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli.
This culture system will facilitate the study of interindividual, functional studies of human intestinal epithelial cells, including host-microbial interactions.
The microbiota is known to modulate the host response to influenza infection through as-yet-unclear mechanisms. We hypothesized that components of the microbiota exert effects through type I ...interferon (IFN), a hypothesis supported by analysis of influenza in a gain-of-function genetic mouse model. Here we show that a microbially associated metabolite, desaminotyrosine (DAT), protects from influenza through augmentation of type I IFN signaling and diminution of lung immunopathology. A specific human-associated gut microbe, Clostridium orbiscindens, produced DAT and rescued antibiotic-treated influenza-infected mice. DAT protected the host by priming the amplification loop of type I IFN signaling. These findings show that specific components of the enteric microbiota have distal effects on responses to lethal infections through modulation of type I IFN.
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