In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multi-cellular 3D structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumor-derived extracellular matrix (ECM), which severely limits use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs such that 3D structures are never embedded in tumor-derived ECM. We also demonstrate that the hydrogel serves as an injectable HIO vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.

b Dysbiosis in the intestinal microbiota of persons with inflammatory bowel disease (IBD) has been described, but there are still varied reports on changes in the abundance of Bifidobacterium and Lactobacillus organisms in patients with IBD. The aim of this investigation was to compare the compositions of mucosa-associated and fecal bacteria in patients with IBD and in healthy controls (HCs). Fecal and biopsy samples from 21 HCs, 21 and 15 Crohn's disease (CD) patients, and 34 and 29 ulcerative colitis (UC) patients, respectively, were analyzed by quantitative real-time PCR targeting the 16S rRNA gene. The bacterial numbers were transformed into relative percentages for statistical analysis. The proportions of bacteria were uniformly distributed along the colon regardless of the disease state. Bifidobacterium was significantly increased in the biopsy specimens of active UC patients compared to those in the HCs (4.6% versus 2.1%, P ‫؍‬ 0.001), and the proportion of Bifidobacterium was significantly higher in the biopsy specimens than in the fecal samples in active CD patients (2.7% versus 2.0%, P ‫؍‬ 0.012). The Lactobacillus group was significantly increased in the biopsy specimens of active CD patients compared to those in the HCs (3.4% versus 2.3%, P ‫؍‬ 0.036). Compared to the HCs, Faecalibacterium prausnitzii was sharply decreased in both the fecal and biopsy specimens of the active CD patients (0.3% versus 14.0%, P < 0.0001 for fecal samples; 0.8% versus 11.4%, P < 0.0001 for biopsy specimens) and the active UC patients (4.3% versus 14.0%, P ‫؍‬ 0.001 for fecal samples; 2.8% versus 11.4%, P < 0.0001 for biopsy specimens). In conclusion, Bifidobacterium and the Lactobacillus group were increased in active IBD patients and should be used more cautiously as probiotics during the active phase of IBD. Butyrate-producing bacteria might be important to gut homeostasis.

SummaryThe current study aimed to understand the developmental mechanisms regulating bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified cues that maintained isolated human fetal lung epithelial bud tip progenitor cells in vitro and induced three-dimensional hPSC-derived organoids with bud tip-like domains. Bud tip-like domains could be isolated, expanded, and maintained as a nearly homogeneous population. Molecular and cellular comparisons revealed that hPSC-derived bud tip-like cells are highly similar to native lung bud tip progenitors. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed, maintained multilineage potential, and successfully engrafted into the airways of immunocompromised mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.

f Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile. C lostridium difficile is an anaerobic, spore-forming bacterium that is the leading cause of infectious nosocomial diarrhea and is responsible for over 14,000 deaths annually (1). Human exposure to C. difficile results in a range of manifestations, from asymptomatic colonization, to diarrhea, to lethal toxic megacolon. Various models have been used to study C. difficile infection (CDI), including in vitro models using transformed cell lines and a variety of in vivo models (2-5). In vitro cell culture models are limited in their ability to recapitulate complexities of the human gastrointestinal tract, and detailed, real-time study of the mucosal epithelium during infection in an animal model is technically challenging.Human intestinal organoids (HIOs) are three-dimensional spheroids of human epithelium generated through directed differentiation of human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HIOs contain both mesenchymal and epithelial tissues that are structurally arranged around a central luminal cavity. The epithelial compartment of the HIO possesses an array of small intestinal cell types, including absorptive enterocytes and secretory Paneth, goblet, and enteroendocrine cells, in addition to Lgr5 ϩ intestinal stem cells (6). HIOs have been used to model features of embryonic development, viral infection, and inflammatory bowel disease (7-9). Due to their similarity to the human gastrointestinal tract, HIOs serve as a tractable and physiologically relevant model of the human intestine.We sought to use HIOs to study the interaction between C. difficile and complex human epit...

Background & Aims Hypoxic inflammation (decreased oxygen tension at sites of inflammation) is a feature of inflammatory bowel disease (IBD). The hypoxia response is mediated by the transcription factors hypoxia-inducible factor (HIF)1α and endothelial PAS domain protein 1 (EPAS1 or HIF2α), which are induced in intestinal tissues of patients with IBD. HIF1α limits intestinal barrier dysfunction, but the role of EPAS1 has not been assessed under conditions of hypoxic inflammation or in models of IBD. Methods Acute colitis was induced by administration of Citrobacter rodentium ordextran sulfate sodium (DSS) to transgenic hypoxia reporter mice (ODD-Luc), mice with conditional overexpression of Epas1 (Epas1LSL/LSL), mice with intestinal epithelium-specific deletion of Epas1 (Epas1ΔIE), or wild-type littermates (controls). Colon tissues from these mice and from patients with ulcerative colitis (UC) or Crohn's disease (CD) were assessed by histologic and immunoblot analyses, immunohistochemistry, and quantitative PCR. Results Levels of hypoxia and EPAS1 were increased in colon tissues of mice following induction of colitis and patients with UC or CD, compared with controls. Epas1ΔIE mice had attenuated colonic inflammation and were protected from DSS-induced colitis. Intestine-specific overexpression of EPAS1, but not HIF-1α, led to spontaneous colitis, increased susceptibility to induction of colitis by C rodentium or DSS, and reduced survival times compared with controls. Disruption of intestinal epithelial EPAS1 attenuated the inflammatory response following administration of DSS or C rodentium, whereas intestine-specific overexpression of EPAS1 increased this response. We found EPAS1 to be a positive regulator of tumor necrosis factor (TNF)α production by the intestinal epithelium. Blocking TNFα completely reduced hypoxia-induced intestinal inflammation. We found EPAS1 to be a positive regulator of tumor necrosis factor (TNF)α production by the intestinal epithelium. Blocking TNFα completely reduced hypoxia-induced intestinal inflammation. Conclusions EPAS1 is a transcription factor that activates mediators of inflammation, such as TNFα, in the intestinal epithelium and promotes development of colitis in mice.