Ontogeny and function of the circadian clock in intestinal organoids.

Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses to TcdB. RNA-Seq analysis shows that ~3-10% of the detectable transcripts are rhythmically expressed in mouse and human enteroids. Remarkably, we observe anti-phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids, and disruption of Rac1 abolishes clock-dependent necrotic cell death responses. Our findings uncover robust functions of circadian rhythms regulating clock-controlled genes in both mouse and human enteroids governing organism-specific, circadian phase-dependent necrotic cell death responses, and lay a foundation for human organ- and disease-specific investigation of clock functions using human organoids for translational applications.

Using Human Induced Pluripotent Stem Cell-Derived Organoids to Identify New Pathologies in Patients With PDX1 Mutations.

BACKGROUND & AIMS: Two patients with homozygous mutations in PDX1 presented with pancreatic agenesis, chronic diarrhea, and poor weight gain, the causes of which were not identified through routine clinical testing. We aimed to perform a deep analysis of the stomach and intestine using organoids derived from induced pluripotent stem cells from PDX1188delC/188delC patients. METHODS: Gastric fundic, antral, and duodenal organoids were generated using induced pluripotent stem cell lines from a PDX1188delC/188delC patient and an isogenic induced pluripotent stem cell line where the PDX1 point mutation was corrected. RESULTS: Patient-derived PDX1188delC/188delC antral organoids exhibited an intestinal phenotype, whereas intestinal organoids underwent gastric metaplasia with significant reduction in enteroendocrine cells. This prompted a re-examination of gastric and intestinal biopsy specimens from both PDX1188delC/188delC patients, which recapitulated the organoid phenotypes. Moreover, antral biopsy specimens also showed increased parietal cells and lacked G cells, suggesting loss of antral identity. All organoid pathologies were reversed upon CRISPR-mediated correction of the mutation. CONCLUSIONS: These patients will now be monitored for the progression of metaplasia and gastrointestinal complications that might be related to the reduced gastric and intestinal endocrine cells. This study demonstrates the utility of organoids in diagnosing uncovered pathologies.

Wnt/ß-catenin promotes gastric fundus specification in mice and humans.

Despite the global prevalence of gastric disease, there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/ß-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium, and that ß-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology, and also represent a new platform for drug discovery.

Intestinal organoids: roadmap to the clinic.

Recent advances in intestinal organoid research, along with encouraging preclinical proof-of-concept studies, have revealed significant therapeutic potential for induced pluripotent stem cell (iPSC)-derived organoids in the healing and replacement of severely injured or diseased bowel (Finkbeiner et al. Biol Open 4: 1462-1472, 2015; Kitano et al. Nat Commun 8: 765, 2017; Cruz-Acuna et al. Nat Cell Biol 19: 1326-1335, 2017). To fully realize the tremendous promise of stem cell organoid-based therapies, careful planning aligned with significant resources and efforts must be devoted demonstrating their safety and efficacy to meet critical regulatory requirements. Early recognition of the inherent preclinical and clinical obstacles that occur with the novel use of pluripotent stem cell-derived products will accelerate their bench-to-bedside translation (Neofytou et al. J Clin Invest 125: 2551-2557, 2015; O'Brien et al. Stem Cell Res Ther 6: 146, 2015; Ouseph et al. Cytotherapy 17: 339-343, 2015). To overcome many of these hurdles, a close and effective collaboration is needed between experts from various disciplines, including basic and clinical research, product development and manufacturing, quality assurance and control, and regulatory affairs. Therefore, the purpose of this article is to outline the critical areas and challenges that must be addressed when transitioning laboratory-based discovery, through an investigational new drug (IND) application to first-in-human clinical trial, and to encourage investigators to consider the required regulatory steps from the earliest stage of the translational process. The ultimate goal is to provide readers with a draft roadmap that they could use while navigating this exciting cell therapy space.

Increased Programmed Death-Ligand 1 is an Early Epithelial Cell Response to Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) is the major risk factor for the development of gastric cancer. Our laboratory has reported that the Sonic Hedgehog (Shh) signaling pathway is an early response to infection that is fundamental to the initiation of H. pylori-induced gastritis. H. pylori also induces programmed death ligand 1 (PD-L1) expression on gastric epithelial cells, yet the mechanism is unknown. We hypothesize that H. pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Shh signaling pathway during infection. To identify the role of Shh signaling as a mediator of H. pylori-induced PD-L1 expression, human gastric organoids generated from either induced pluripotent stem cells (HGOs) or tissue (huFGOs) were microinjected with bacteria and treated with Hedgehog/Gli inhibitor GANT61. Gastric epithelial monolayers generated from the huFGOs were also infected with H. pylori and treated with GANT61 to study the role of Hedgehog signaling as a mediator of induced PD-1 expression. A patient-derived organoid/autologous immune cell co-culture system infected with H. pylori and treated with PD-1 inhibitor (PD-1Inh) was developed to study the protective mechanism of PD-L1 in response to bacterial infection. H. pylori significantly increased PD-L1 expression in organoid cultures 48 hours post-infection when compared to uninfected controls. The mechanism was cytotoxic associated gene A (CagA) dependent. This response was blocked by pretreatment with GANT61. Anti-PD-L1 treatment of H. pylori infected huFGOs, co-cultured with autologous patient cytotoxic T lymphocytes and dendritic cells, induced organoid death. H. pylori-induced PD-L1 expression is mediated by the Shh signaling pathway within the gastric epithelium. Cells infected with H. pylori that express PD-L1 may be protected from the immune response, creating premalignant lesions progressing to gastric cancer.

Durability of rifaximin response in hepatic encephalopathy.

GOALS: To evaluate the durability of the response to rifaximin for treatment of hepatic encephalopathy (HE). BACKGROUND: The nonsystemic antibiotic rifaximin has been approved for maintenance of HE remission, and several studies have indicated the efficacy of rifaximin for acute HE; however, the duration of therapeutic response for >6 months remains unknown. STUDY: Medical records of patients with cirrhosis who received rifaximin maintenance therapy for HE between January 2004 and May 2009 were reviewed. Model for end-stage liver disease (MELD) scores were obtained every 3 months during therapy. RESULTS: Of 203 patients with HE (Conn score ≥2), 149 received rifaximin monotherapy (400 to 1600 mg/d) and 54 received rifaximin (600 to 1200 mg/d) and lactulose (90 mL/d) dual therapy. Maintenance of HE remission for 1 year occurred in 81% and 67% of patients who received rifaximin monotherapy and rifaximin and lactulose dual therapy, respectively. Patient populations with a baseline mean MELD score ≤20 had few overt HE events, suggesting increased response to rifaximin in these patients. CONCLUSIONS: Rifaximin is effective for the management of HE in patients with cirrhosis, particularly in populations with MELD scores ≤20. Additional studies are needed to investigate the potential association between MELD scores and the efficacy of HE treatments.

Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation.

A major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

An integrated microfluidic bubble pocket for long-term perfused three-dimensional intestine-on-a-chip model.

Perfused three-dimensional (3D) cultures enable long-term in situ growth and monitoring of 3D organoids making them well-suited for investigating organoid development, growth, and function. One of the limitations of this long-term on-chip perfused 3D culture is unintended and disruptive air bubbles. To overcome this obstacle, we invented an imaging platform that integrates an innovative microfluidic bubble pocket for long-term perfused 3D culture of gastrointestinal (GI) organoids. We successfully applied 3D printing technology to create polymer molds that cast polydimethylsiloxane (PDMS) culture chambers in addition to bubble pockets. Our developed platform traps unintended, or induced, air bubbles in an integrated PDMS pocket chamber, where the bubbles diffuse out across the gas permeable PDMS or an outlet tube. We demonstrated that our robust platform integrated with the novel bubble pocket effectively circumvents the development of bubbles into human and mouse GI organoid cultures during long-term perfused time-course imaging. Our platform with the innovative integrated bubble pocket is ideally suited for studies requiring long-term perfusion monitoring of organ growth and morphogenesis as well as function.

Generation of human antral and fundic gastric organoids from pluripotent stem cells.

The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs) into human gastric organoids (hGOs) that recapitulate both domains. Both protocols begin with the differentiation of hPSCs into definitive endoderm (DE) using activin A, followed by the generation of free-floating 3D posterior foregut spheroids using FGF4, Wnt pathway agonist CHIR99021 (CHIR), BMP pathway antagonist Noggin, and retinoic acid. Embedding spheroids in Matrigel and continuing 3D growth in epidermal growth factor (EGF)-containing medium for 4 weeks results in antral hGOs (hAGOs). To obtain fundic hGOs (hFGOs), spheroids are additionally treated with CHIR and FGF10. Induced differentiation of acid-secreting parietal cells in hFGOs requires temporal treatment of BMP4 and the MEK inhibitor PD0325901 for 48 h on protocol day 30. In total, it takes ~34 d to generate hGOs from hPSCs. To date, this is the only approach that generates functional human differentiated gastric cells de novo from hPSCs.

Human stomach-on-a-chip with luminal flow and peristaltic-like motility.

Current in vitro approaches and animal models have critical limitations for modeling human gastrointestinal diseases because they may not properly represent multicellular human primary tissues. Therefore, there is a need for model platforms that recapitulate human in vivo development, physiology, and disease processes to validate new therapeutics. One of the major steps toward this goal was the generation of three-dimensional (3D) human gastric organoids (hGOs) via the directed differentiation of human pluripotent stem cells (hPSCs). The normal functions and diseases of the stomach occur in the luminal epithelium, however accessing the epithelium on the inside of organoids is challenging. We sought to develop a bioengineered platform to introduce luminal flow through hGOs to better model in vivo gastric functions. Here, we report an innovative microfluidic imaging platform housing hGOs with peristaltic luminal flow in vitro. This human stomach-on-a-chip allows robust, long-term, 3D growth of hGOs with the capacity for luminal delivery via a peristaltic pump. Organoids were cannulated and medium containing fluorescent dextran was delivered through the lumen using a peristaltic pump. This system also allowed us to rhythmically introduce stretch and contraction to the organoid, reminiscent of gastric motility. Our platform has the potential for long-term delivery of nutrients or pharmacological agents into the gastric lumen in vitro for the study of human gastric physiology, disease modeling, and drug screening, among other possibilities.