Pluripotent Stem Cell-derived Intestinal Organoids Research Articles

DOP88 Lipids drive myofibroblast activation and the epithelial-mesenchymal transition process in Crohn’s Disease

Abstract Background Fibrostenosis represents one of the major clinical complications of Crohn’s Disease (CD). However, the molecular mechanisms underlying intestinal fibrogenesis remain poorly understood and anti-fibrotic therapy has not yet been developed. Growing evidences supports that oxidative stress and the epithelial-mesenchymal transition (EMT) process contribute to the development and progression of fibrosis. Here, we assessed the role of lipids in fibrogenesis and whether mitochondria dysfunction can support fibrosis formation. Methods Spatial metabolomics analysis and adipophilin (ADFP) staining were performed on surgical specimens from fibrotic ileum and the surrounding region (healthy) derived from CD patients. Fibroblasts isolated from fibrotic and healthy ileum and human induced pluripotent stem cell-derived intestinal organoids (iHOs) were treated for 1-3 weeks with lipid mix (2%) and analyzed by RNA sequencing. Pathways significantly modulated were validated by RT-PCR, WB, FACS analysis, and proliferative, migratory and contraction assays, and mitochondrial functions by Seahorse analyzer. Results Spatial metabolomics revealed a significant accumulation of lipids characterized by very long-chain fatty acids in the fibrotic area compared to surrounding healthy regions, which was further confirmed by ADFP staining. RNA-seq analysis showed overlapping dysregulated pathways between healthy fibroblasts stimulated with lipids and fibrotic fibroblasts, in particular genes related to cell cycle, cytoskeletal reorganization, migration, contractility and cell metabolism (oxidative stress) which were further confirmed by functional assays. Among the genes related to oxidative stress, lipid stimulation significantly decreased NFE2L2 and promoted an increase in CPT1A, gene encoding for a fatty oxidation mediator. Moreover, lipid-stimulated fibroblasts showed a reduction in mitochondrial respiration, particularly basal and maximal respiration, and spare respiratory capacity by Seahorse analysis. On the other hand, long exposure of iHOs to lipids promoted the EMT process through the upregulation of TWIST, ZEB, VIM, and S100a4. Conclusion Lipid stimulation induced myofibroblast activation through oxidative stress and alteration of mitochondrial metabolism, and increased the expression of EMT transcription factors in intestinal epithelial cells (iHOs) that may support fibroblast-mediated pro-fibrotic activity. These data indicate that regulators of fatty acid oxidation involved in the fibrotic process may represent new potential targets for the treatment of fibrostenotic CD patients.

ECCO Grant Dissecting the role of S1PR1 modulator in the mucosal regeneration of Ulcerative Colitis

Abstract Background and Aims Ozanimod, a potent sphingosine 1-phosphate (S1P) 1/5 receptor agonist, first approved for the treatment of adults with moderately to severely active UC in the USA and in the EU, has showed promising results in terms of both tolerability and efficacy reducing inflammatory processes and inducing disease remission. The selectivity of Ozanimod for S1PR1 is 27 times that for S1PR5. Therefore, is plausible that the beneficial effects of Ozanimod are mediated by S1PR1, which is constitutively expressed not only by immune cells, but also by epithelial, endothelial, and stromal cells. However, its function on non-immune cells has been neglected. Based on preliminary data demonstrating a faster recovery of mucosal damage in mice with a conditional deletion of S1PR1 in the epithelia and endothelia compartments, our hypothesis is that the inactivation of S1PR1 not only inhibits immune cell trafficking, but also promotes tissue repair. Based on this background, the main objectives will be to exploit the Ozanimod effects during the process of mucosa healing, and to investigate the molecular pathways activated by Ozanimod on epithelial and endothelial cells. Methods Pre-clinical validation of Ozanimod, in vivo model of UC in association with conditional knockout mice for S1PR1 on epithelial and endothelial cells, stimulation of primary endothelial cells from UC patients and human induced pluripotent stem cell-derived intestinal organoids (iHOs) will be used to address our aims. Transcriptomics, functional assay, immune characterization, WB validation will be performed. Anticipated Impact In short and medium term, we will improve the knowledge on the role of S1PR1 in the processes of mucosal healing and identify the molecular pathways involved in the response to the mucosal damage mediated by the ozanimod in non-immune cells. The results of this study may improve the treatment options for UC patients.

A Novel Lithocholic Acid Derivative Upregulates Detoxification-Related Genes in Human Induced Pluripotent Stem Cell-Derived Intestinal Organoids.

Vitamin D is a fat-soluble micronutrient that plays essential roles in a range of biological processes, including cell proliferation, inflammation, and metabolism. In this study, we investigated the effects of a novel synthetic lithocholic acid derivative with vitamin D activity (Dcha-20) on pharmacokinetic gene expression in human induced pluripotent stem cell-derived intestinal organoids. Compared with vitamin D3 treatment, Dcha-20 was found to upregulate the expression and enzyme activity of the drug-metabolizing enzyme CYP3A4, an indicator of intestinal functional maturation. In addition, Dcha-20 specifically increased expression levels of the xenobiotic detoxification enzyme UGT1A and excretion transporter MRP2. These results suggest that Dcha-20 promotes activity of the intrinsic defense system of the intestinal epithelium.

Effects of human induced pluripotent stem cell-derived intestinal organoids on colitis-model mice

IntroductionUlcerative colitis (UC) is an inflammatory bowel disease characterized by repeated remissions and relapses. Immunosuppressive drugs have facilitated the induction and maintenance of remission in many patients with UC. However, immunosuppressive drugs cannot directly repair impaired intestinal mucosa and are insufficient for preventing relapse. Therefore, new treatment approaches to repair the damaged epithelium in UC have been attempted through the transplantation of intestinal organoids, which can be differentiated into mucosa by embedding in Matrigel, generated from patient-derived intestinal stem cells. The method, however, poses the challenge of yielding sufficient cells for UC therapy, and patient-derived cells might already have acquired pathological changes. In contrast, human induced pluripotent stem (iPS) cells generated from healthy individuals are infinitely proliferated and can be differentiated into target cells. Recently developed human iPS cell-derived intestinal organoids (HIOs) aim to generate organoids that closely resemble the adult intestine. However, no study till date has reported HIOs injected into in vivo inflammatory models, and it remains unclear whether HIOs with cells that closely resemble the adult intestine or with intestinal stem cells retain the better ability to repair tissue in colitis.MethodsWe generated two types of HIOs via suspension culture with and without small-molecule compounds: HIOs that include predominantly more intestinal stem cells [HIO (A)] and those that include predominantly more intestinal epithelial and secretory cells [HIO (B)]. We examined whether the generated HIOs engrafted in vivo and compared their ability to accelerate recovery of the damaged tissue.ResultsFindings showed that the HIOs expressed intestinal-specific markers such as caudal-type homeobox 2 (CDX2) and villin, and HIOs engrafted under the kidney capsules of mice. We then injected HIOs into colitis-model mice and found that the weight and clinical score of the mice injected with HIO (A) recovered earlier than that of the mice in the sham group. Further, the production of mucus and the expression of cell proliferation markers and tight junction proteins in the colon tissues of the HIO (A) group were restored to levels similar to those observed in healthy mice. However, neither HIO (A) nor HIO (B) could be engrafted into the colon.ConclusionsEffective cell therapy should directly repair tissue by engraftment at the site of injury. However, the difference in organoid property impacting the rate of tissue repair in transplantation without engraftment observed in the current study should be considered a critical consideration in the development of regenerative medicine using iPS-derived organoids.

Suspension culture of human induced pluripotent stem cell-derived intestinal organoids using natural polysaccharides

Currently, there are many challenges in the culture of human induced pluripotent stem (iPS) cell-derived intestinal organoids (HIOs) for use in drug discovery, disease research, and regenerative medicine. For example, the main culture method, embedding culture, makes industrial large-scale culture difficult, and Matrigel, which is used for almost all HIO cultures, is not respected for its application in regenerative medicine. To overcome these challenges, we herein propose a new culture method using low concentrations of natural polysaccharides in a suspension culture. In the present study, five natural polysaccharides free from heterologous animal-derived components were used, and HIOs were successfully cultured in suspension with FP001 and FP003, which are microbial exopolysaccharide analogs of gellan gum. The fabricated HIOs were similar to living intestinal tracts with respect to their gene expression, microstructure, and protein expression. The observed activities of the drug metabolizing enzymes and drug transporters in the generated HIOs suggested that they have pharmacokinetic functions. We believe that suspension culture of HIOs using FP001 or FP003 can be widely applied to not only drug discovery research but also disease research and regenerative medicine.

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids.

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Regional specification in the intestine is driven by multiple developmental pathways, including the bone morphogenetic protein (BMP) pathway. Based on normal regional specification, a method to generate human colonic organoids (HCOs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hES) and induced pluripotent stem cells (iPSCs), was developed. A three-day induction of BMP signaling sufficiently patterns mid/hindgut tube cultures into special AT-rich sequence-binding protein 2 (SATB2)-expressing HCOs containing all of the main epithelial cell types present in human colon as well as co-developing mesenchymal cells. Omission of BMP (or addition of the BMP inhibitor NOGGIN) during this critical patterning period resulted in the formation of human intestinal organoids (HIOs). HIOs and HCOs morphologically and molecularly resemble human developing small intestine and colon, respectively. Despite the utility of HIOs and HCOs for studying human intestinal development, the generation of HIOs and HCOs is challenging. This paper presents methods for generating, maintaining, and characterizing HIOs and HCOs. In addition, the critical steps in the protocol and troubleshooting recommendations are provided.

Human Pluripotent Stem Cell-Derived Intestinal Organoids Model SARS-CoV-2 Infection Revealing a Common Epithelial Inflammatory Response.

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to coronavirus disease 2019 (COVID-19) usually results in respiratory disease, but extrapulmonary manifestations are of major clinical interest. Intestinal symptoms of COVID-19 are present in a significant number of patients, and include nausea, diarrhea, and viral RNA shedding in feces. Human induced pluripotent stem cell-derived intestinal organoids (HIOs) represent an inexhaustible cellular resource that could serve as a valuable tool to study SARS-CoV-2 as well as other enteric viruses that infect the intestinal epithelium. Here, we report that SARS-CoV-2 productively infects both proximally and distally patterned HIOs, leading to the release of infectious viral particles while stimulating a robust transcriptomic response, including a significant upregulation of interferon-related genes that appeared to be conserved across multiple epithelial cell types. These findings illuminate a potential inflammatory epithelial-specific signature that may contribute to both the multisystemic nature of COVID-19 as well as its highly variable clinical presentation.

Generation of Budding-Like Intestinal Organoids from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem (iPS) cell-derived intestinal organoids have low invasiveness; however, the current differentiation method does not reflect the crypt-villus-like structure due to structural immaturity. Here, we generated budding-like organoids that formed epithelial tissue-like structures and had the characteristics of the mature small intestine from human iPS cells. They showed a high expression of drug transporters and induced the expression of cytochrome P450 3A4 and P-glycoprotein. When treated with tumor necrosis factor-α and/or transforming growth factor-β, the budding-like organoids replicated the pathogenesis of mucosal damage or intestinal fibrosis. Upon dissociation and seeding on cell culture inserts, the organoids retained intestinal characteristics, forming polarized intestinal folds with approximately 400 Ω × cm2 transepithelial electrical resistance. This novel method has great potential for disease modeling and drug screening applications.

Human Pluripotent Stem Cell-Derived Organoids as a Model of Intestinal Xenobiotic Metabolism

Background: The human intestine is the site of absorption and first-pass metabolism for oral intake. Assessment of absorption, distribution, metabolism, excretion, and toxicity (ADMET) of xenobiotics has transformed the understanding of in vivo pharmacology. However, these processes are difficult torecapitulate in vitro. Objective: We have developed a simple protocol for the generation of mature functional intestinal organoids from human pluripotent stem cells (hPSCs)under xenogeneic-free conditions. We sought to characterize transcription level in drug transporters and metabolism and evaluate CYP3A4 catalytic function of the organoids. Methods: Human pluripotent stem cell-derived intestinal organoids were generated and evaluated the expression of drug transporters and metabolizing enzymes. We examined the induction of CYP3A4 and ABCB1 gene expression in the organoids. Furthermore, we analyzed the CYP3A4 enzyme activity of the organoids by the p450-Glo CYP3A4 assay kit with luciferin isopropyl acetal. Results: Stem cell-derived intestinal organoids had an outward polarized intestinal epithelial layer and showed similar expression levels of drug transporters and metabolism genes as the adult healthy intestine. They also exhibited CYP3A4 enzymatic function in vitro. Conclusion: This model provides a novel platform for pharmacological testing and can enhance human ADMET studies in drug development.

Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.

Application of Human Induced Pluripotent Stem Cell-Derived Intestinal Organoids as a Model of Epithelial Damage and Fibrosis in Inflammatory Bowel Disease.

Inflammatory bowel disease, which typically manifests as Crohn's disease and ulcerative colitis, is caused by the abnormal production of cytokines such as tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β. These cytokines damage intestinal epithelial cells and trigger fibrosis, respectively, for which the current in vitro models have many limitations. Therefore, we tested whether human induced pluripotent stem cell-derived intestinal organoids (HiOs) can mimic inflammatory bowel disease (IBD), and whether such a model is suitable for drug screening. HiOs were treated with TNF-α and TGF-β to construct mucosal damage and fibrosis models. TNF-α diminished the mRNA expression of intestinal epithelial cell and goblet cell markers in HiOs. TNF-α also induced epithelial cell damage and degradation of tight junctions but not in the presence of infliximab, an antibody used in the clinic to deplete TNF-α. Furthermore, permeation of the non-absorbable marker FD-4 was observed in HiOs treated with TNF-α or ethylene glycol tetraacetic acid (EGTA), but not in the presence of infliximab. In contrast, TNF-α and TGF-β induced mRNA expression of mesenchymal and fibrosis markers, as well as epithelial-mesenchymal transition. SB431542, a TGF-β inhibitor, significantly reversed these events. The data indicate that HiOs mimic mucosal damage and fibrosis due to IBD and are thus suitable models for drug screening.

Evaluating the usefulness of human-induced pluripotent stem cell-derived intestinal organoids in drug discovery research

Evaluating the usefulness of human-induced pluripotent stem cell-derived intestinal organoids in drug discovery research

Interleukin-2 induces the in vitro maturation of human pluripotent stem cell-derived intestinal organoids

Human pluripotent stem cell (hPSC)-derived intestinal organoids (hIOs) form 3D structures organized into crypt and villus domains, making them an excellent in vitro model system for studying human intestinal development and disease. However, hPSC-derived hIOs still require in vivo maturation to fully recapitulate adult intestine, with the mechanism of maturation remaining elusive. Here, we show that the co-culture with human T lymphocytes induce the in vitro maturation of hIOs, and identify STAT3-activating interleukin-2 (IL-2) as the major factor inducing maturation. hIOs exposed to IL-2 closely mimic the adult intestinal epithelium and have comparable expression levels of mature intestinal markers, as well as increased intestine-specific functional activities. Even after in vivo engraftment, in vitro-matured hIOs retain their maturation status. The results of our study demonstrate that STAT3 signaling can induce the maturation of hIOs in vitro, thereby circumventing the need for animal models and in vivo maturation.

Efficient Generation of Cynomolgus Monkey Induced Pluripotent Stem Cell-Derived Intestinal Organoids with Pharmacokinetic Functions.

In preclinical studies, the cynomolgus monkey (CM) model is frequently used to predict the pharmacokinetics of drugs in the human small intestine, because of its evolutionary closeness to humans. Intestinal organoids that mimic the intestinal tissue have attracted attention in regenerative medicine and drug development. In this study, we generated intestinal organoids from CM induced pluripotent stem (CMiPS) cells and analyzed their pharmacokinetic functions. CMiPS cells were induced into the hindgut; then, the cells were seeded on microfabricated culture vessel plates to form spheroids. The resulting floating spheroids were differentiated into intestinal organoids in a medium containing small-molecule compounds. The mRNA expression of intestinal markers and pharmacokinetic-related genes was markedly increased in the presence of small-molecule compounds. The organoids possessed a polarized epithelium and contained various cells constituting small intestinal tissues. The intestinal organoids formed functional tight junctions and expressed drug transporter proteins. In addition, in the organoids generated, cytochrome P450 3A8 (CYP3A8) activity was inhibited by the specific inhibitor ketoconazole and was induced by rifampicin. Therefore, in the present work, we successfully generated intestinal organoids, with pharmacokinetic functions, from CMiPS cells using small-molecule compounds.

Efficient generation of human and cynomolgus monkey induced pluripotent stem cell-derived intestinal organoids with pharmacokinetic functions

Efficient generation of human and cynomolgus monkey induced pluripotent stem cell-derived intestinal organoids with pharmacokinetic functions

A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids.

SummaryGut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC)-derived intestinal organoids involving four methodological advances. (1) We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2) We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3) Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4) We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

Reciprocal Inflammatory Signaling Between Intestinal Epithelial Cells and Adipocytes in the Absence of Immune Cells

Visceral fat accumulation as observed in Crohn's disease and obesity is linked to chronic gut inflammation, suggesting that accumulation of gut adipocytes can trigger local inflammatory signaling. However, direct interactions between intestinal epithelial cells (IECs) and adipocytes have not been investigated, in part because IEC physiology is difficult to replicate in culture. In this study, we originally prepared intact, polarized, and cytokine responsive IEC monolayers from primary or induced pluripotent stem cell-derived intestinal organoids by simple and repeatable methods. When these physiological IECs were co-cultured with differentiated adipocytes in Transwell, pro-inflammatory genes were induced in both cell types, suggesting reciprocal inflammatory activation in the absence of immunocompetent cells. These inflammatory responses were blocked by nuclear factor-κB or signal transducer and activator of transcription 3 inhibition and by anti-tumor necrosis factor- or anti-interleukin-6-neutralizing antibodies. Our results highlight the utility of these monolayers for investigating IEC biology. Furthermore, this system recapitulates the intestinal epithelium–mesenteric fat signals that potentially trigger or worsen inflammatory disorders such as Crohn's disease and obesity-related enterocolitis.

Growth hormone is permissive for neoplastic colon growth

Growth hormone (GH) excess in acromegaly is associated with increased precancerous colon polyps and soft tissue adenomas, whereas short-stature humans harboring an inactivating GH receptor mutation do not develop cancer. We show that locally expressed colon GH is abundant in conditions predisposing to colon cancer and in colon adenocarcinoma-associated stromal fibroblasts. Administration of a GH receptor (GHR) blocker in acromegaly patients induced colon p53 and adenomatous polyposis coli (APC), reversing progrowth GH signals. p53 was also induced in skin fibroblasts derived from short-statured humans with mutant GHR. GH-deficient prophet of pituitary-specific positive transcription factor 1 (Prop1)(-/-) mice exhibited induced colon p53 levels, and cross-breeding them with Apc(min+/-) mice that normally develop intestinal and colon tumors resulted in GH-deficient double mutants with markedly decreased tumor number and size. We also demonstrate that GH suppresses p53 and reduces apoptosis in human colon cell lines as well as in induced human pluripotent stem cell-derived intestinal organoids, and confirm in vivo that GH suppresses colon mucosal p53/p21. GH excess leads to decreased colon cell phosphatase and tensin homolog deleted on chromosome 10 (PTEN), increased cell survival with down-regulated APC, nuclear β-catenin accumulation, and increased epithelial-mesenchymal transition factors and colon cell motility. We propose that GH is a molecular component of the "field change" milieu permissive for neoplastic colon growth.