Protocol to generate large human intestinal organoids using a rotating bioreactor.

Current techniques for producing induced-pluripotent-stem-cell-derived mid/hindgut spheroids have faced major hurdles in consistency and reproducibility. Here, we present a protocol that uses mid/hindgut cells to generate homogeneous spheroids that subsequently mature into human intestinal organoids (HIOs). We describe steps for stepwise differentiation and spheroid formation using a 96-well plate. We then detail cell maturation in a suspended state and the implementation of a rotational bioreactor platform to maximize the culture efficiency of larger HIOs. For complete details on the use and execution of this protocol, please refer to Takahashi et al.1.

Organoids transplantation as a new modality to design epithelial signature to create a membrane-protective sulfomucin-enriched segment.

BACKGROUND: The organoids therapy for ulcerative colitis (UC) is under development. It is important to dissect how the engrafted epithelium can provide benefits for overcoming the vulnerability to inflammation. We mainly focused on the deliverability of sulfomucin, which is reported to play an important role in epithelial function. METHODS: We analyzed each segment of colon epithelium to determine differences in sulfomucin production in both mice and human. Subsequently, we transplanted organoids established from sulfomucin-enriched region into the injured recipient epithelium following dextran sulfate sodium-induced colitis and analyzed the engrafted epithelium in mouse model. RESULTS: In human normal colon, sulfomucin production was increased in proximal colon, whereas it was decreased in the inflammatory region of UC. In murine colon epithelium, increased sulfomucin production was found in cecum compared to distal small intestine and proximal colon. RNA sequencing analysis revealed that several key genes associated with sulfomucin production such as Papss2 and Slc26a1 were enriched in isolated murine cecum crypts. Then we established murine cecum organoids and transplanted them into the injured epithelium of distal colon. Although the expression of sulfomucin was temporally decreased in cecum organoids, its secretion was restored again in the engrafted patches after transplantation. Finally, we verified a part of mechanisms controlling sulfomucin production in human samples. CONCLUSION: This study illustrated the deliverability of sulfomucin in the disease-relevant grafting model to design sulfomucin-producing epithelial units in severely injured distal colon. The current study is the basis for the better promotion of organoids transplantation therapy for refractory UC.

Adrenomedullin alleviates mucosal injury in experimental colitis and increases claudin-4 expression in the colonic epithelium.

Adrenomedullin (AM) is a peptide with pleiotropic physiological functions that attenuates intestinal mucosal inflammation. However, the mechanism underpinning mucosal protection by AM is not fully understood, and its effect on intestinal epithelial cells remains unclear. Here, we investigated the effects of AM on junctional molecules in primary-cultured murine intestinal epithelial cells and discovered that AM upregulates claudin-4 expression. In a mouse model of dextran sulfate sodium-induced colitis, AM administration also enhanced claudin-4 expression and accelerated mucosal regeneration. Furthermore, AM reversed TNFα-mediated downregulation of claudin-4 and loss of cell-cell adhesion of the HCT116 human intestinal epithelial cell line in vitro. These results indicate that AM may enhance intestinal epithelial integrity by upregulating claudin-4 expression.

Suspension culture in a rotating bioreactor for efficient generation of human intestinal organoids.

Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSCs) hold great promise for translational medical applications. A common method to obtain HIOs has been to harvest floating hindgut spheroids arising from hPSCs. As this technique is elegant but burdensome due to the complex protocol and line-to-line variability, a more feasible method is desired. Here, we establish a robust differentiation method into suspension-cultured HIOs (s-HIOs) by seeding dissociated cells on a spheroid-forming plate. This protocol realizes the reliable generation of size-controllable spheroids. Under optimized conditions in a rotating bioreactor, the generated spheroids quickly grow and mature into large s-HIOs with supporting mesenchyme. Upon mesenteric transplantation, s-HIOs further mature and develop complex tissue architecture in vivo. This method demonstrates that intestinal tissue can be generated from iPSC-derived HIOs via suspension induction and bioreactor maturation, establishing a reliable culture platform with wide applications in regenerative medicine.

Collagen type I-mediated mechanotransduction controls epithelial cell fate conversion during intestinal inflammation.

BACKGROUND: The emerging concepts of fetal-like reprogramming following tissue injury have been well recognized as an important cue for resolving regenerative mechanisms of intestinal epithelium during inflammation. We previously revealed that the remodeling of mesenchyme with collagen fibril induces YAP/TAZ-dependent fate conversion of intestinal/colonic epithelial cells covering the wound bed towards fetal-like progenitors. To fully elucidate the mechanisms underlying the link between extracellular matrix (ECM) remodeling of mesenchyme and fetal-like reprogramming of epithelial cells, it is critical to understand how collagen type I influence the phenotype of epithelial cells. In this study, we utilize collagen sphere, which is the epithelial organoids cultured in purified collagen type I, to understand the mechanisms of the inflammatory associated reprogramming. Resolving the entire landscape of regulatory networks of the collagen sphere is useful to dissect the reprogrammed signature of the intestinal epithelium. METHODS: We performed microarray, RNA-seq, and ATAC-seq analyses of the murine collagen sphere in comparison with Matrigel organoid and fetal enterosphere (FEnS). We subsequently cultured human colon epithelium in collagen type I and performed RNA-seq analysis. The enriched genes were validated by gene expression comparison between published gene sets and immunofluorescence in pathological specimens of ulcerative colitis (UC). RESULTS: The murine collagen sphere was confirmed to have inflammatory and regenerative signatures from RNA-seq analysis. ATAC-seq analysis confirmed that the YAP/TAZ-TEAD axis plays a central role in the induction of the distinctive signature. Among them, TAZ has implied its relevant role in the process of reprogramming and the ATAC-based motif analysis demonstrated not only Tead proteins, but also Fra1 and Runx2, which are highly enriched in the collagen sphere. Additionally, the human collagen sphere also showed a highly significant enrichment of both inflammatory and fetal-like signatures. Immunofluorescence staining confirmed that the representative genes in the human collagen sphere were highly expressed in the inflammatory region of ulcerative colitis. CONCLUSIONS: Collagen type I showed a significant influence in the acquisition of the reprogrammed inflammatory signature in both mice and humans. Dissection of the cell fate conversion and its mechanisms shown in this study can enhance our understanding of how the epithelial signature of inflammation is influenced by the ECM niche.

Development of Long-Acting Human Adrenomedullin Fc-Fusion Proteins.

(1) Background: Human adrenomedullin (hAM) is a hypotensive peptide hormone that exerts powerful anti-inflammatory effects. AM also had therapeutic effects in various animal experimental models of disease. However, treatment required continuous administration as the half-life of native AM is short in blood. To resolve this, we developed four human IgG1 and IgG4 Fc-fusion proteins containing full-length hAM or hAM residues 6-52. (2) Methods: We used mammalian cells to produce recombinant Fc-AM derivatives and tested the pharmacokinetics and biological activity of Fc-AM. (3) Results: We developed four Fc-fusion AMs (Fc-AM), which are long-acting AM derivatives in mammalian cells. Fc-AM had a prolonged half-life in blood and retained its ability to bind to the AM1 receptor. Fc-AM (6-52) induced higher cAMP levels for the receptor than Fc-AM. After the administration of IgG1-AM (6-52) or IgG4-AM (6-52) to rats, tissue transfer to the kidney and small intestine was observed. In addition, treatment with IgG4-AM (6-52) inhibited blood pressure increase in spontaneously hypertensive rats. (4) Conclusions: Fc-AM produced from mammalian cells can be easily prepared and might be an effective novel therapeutic agent.

Zranb1-mutant mice display abnormal colonic mucus production and exacerbation of DSS-induced colitis.

Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1C443S/C443S) mice using the CRISPR/Cas9 system. Zranb1C443S/C443S mice exhibited decreased mRNA expression and MUC2 production. Colonic organoids from Zranb1C443S/C443S mice displayed decreased Muc2 mRNA expression following differentiation into goblet cells. Finally, we analyzed dextran sulfate sodium-induced colitis to understand ZRANB1's role in intestinal inflammation. Zranb1C443S/C443S mice with colitis exhibited significant weight loss, reduced colon length, and worsening clinical and pathological scores, indicating that ZRANB1 contributes to intestinal homeostasis. Together, these results suggest that ZRANB1 regulates MUC2 expression and intestinal inflammation, which may help elucidating the pathogenesis of inflammatory bowel disease and developing new therapeutics targeting ZRANB1.

The usefulness of plasma levels of mature and total adrenomedullin as biomarkers indicating the magnitude of surgical stress responses: A single-center, prospective, observational study.

BACKGROUND AND AIM: Adrenomedullin (AM), a vasodilatory peptide, is known for its pleiotropic actions. AM levels are increased under inflammatory conditions such as sepsis and can be useful as a prognostic biomarker. However, there are only a few reports on the physiological actions of AM in the perioperative period. The aim of this single-center, prospective, and observational study was to investigate the changes in the plasma levels of mature AM (mAM) and total AM (tAM) observed during the perioperative period. In addition, we aimed to determine the association between each AM level and immune-inflammatory parameters to explore the usefulness of AM as a biomarker of the magnitude of surgical stress responses. METHODS: The levels of both mAM and tAM, in addition to the levels of presepsin, interleukin-6, procalcitonin, white blood cell, and C-reactive protein, were measured in blood samples obtained during the perioperative period. Other laboratory data, including sequential organ failure assessment (SOFA) and acute physiology and chronic health evaluation (APACHE) II scores, were obtained from individual clinical records. Correlations between each AM and clinical parameters were determined using Spearman's rank correlation. P<0.05 were considered statistically significant. RESULTS: One hundred and twenty-three perioperative patients scheduled for three types of surgical procedures, including cardiopulmonary bypass surgery, abdominal surgery, and cervical laminoplasty, were included in this study. There was a moderate to strong correlation between each AM and immune-inflammatory parameters, SOFA score, and APACHE II score, as related to surgical trauma. Specifically, the strongest correlation was observed between each AM and SOFA score. CONCLUSIONS: These findings suggest that plasma AM levels may represent the most important inflammatory mediators that are evident in surgical stress responses. RELEVANCE FOR PATIENTS: Since the levels of both tAM and mAM show the same trend, mAM and tAM may be equally used as biomarkers for the evaluation of the physiological status of surgical patients. TRIAL REGISTRATION: This observational study was retrospectively registered with Japanese Clinical Trial Registry "UMIN-CTR" on March 19, 2018, and was given a trial ID number UMIN000031792.

Development of a novel human adrenomedullin derivative: human serum albumin-conjugated adrenomedullin.

Adrenomedullin is a biologically active peptide with multiple functions. Here, we have developed a novel human serum albumin-adrenomedullin (HSA-AM) conjugate, which was synthesized by the covalent attachment of a maleimide derivative of adrenomedullin to the 34th cysteine residue of HSA via a linker. Denaturing gel electrophoresis and western blotting for HSA-AM yielded a single band with adrenomedullin immunoreactivity at the position corresponding to a molecular weight (MW) of 73 kDa. Following gel-filtration chromatography, the purified HSA-AM showed a single main peak corresponding with an MW of 73 kDa, indicating that HSA-AM is a monomer. Both adrenomedullin and HSA-AM stimulated the intracellular accumulation of cyclic AMP (cAMP) in HEK-293 cells stably expressing the adrenomedullin 1 receptor. The pEC50 values for adrenomedullin and HSA-AM were 8.660 and 7.208 (equivalent to 2.19 and 61.9 nM as EC50), respectively. The bioavailability of HSA-AM compared with that of adrenomedullin was much improved after subcutaneous administration in the rat, which was probably due to the superior resistance of HSA-AM towards endogenous proteases and its reduced clearance from the blood. HSA-AM may be a promising drug candidate for clinical application.

11-Ketotestosterone is a major androgen produced in porcine adrenal glands and testes.

Porcine steroid hormone profiles have some unique characteristics. We previously studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs). To investigate porcine steroidogenesis, we induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. Using cAMP, adenovirus-mediated introduction of steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP) induced the differentiation of PSPA cells into sex steroid-producing cells. Introducing SF-1/Ad4BP also induced the aldo-keto reductase 1C1 (AKR1C1) gene. Porcine AKR1C1 had 17ß-hydroxysteroid dehydrogenase activity, which converts androstenedione and 11-ketoandrostenedione into testosterone (T) and 11-ketotestosteorne (11KT). Furthermore, differentiated cells expressed hydroxysteroid 11ß-dehydrogenase 2 (HSD11B2) and produced 11KT. HSD11B2 was expressed in testicular Leydig cells and the adrenal cortex. 11KT was present in the plasma of both immature male and female pigs, with slightly higher levels in the male pigs. T levels were much higher in the male pigs. It is noteworthy that in the female pigs, the 11KT levels were >10-fold higher than the T levels. However, castration altered the 11KT and T plasma profiles in the male pigs to near those of the females. 11KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11KT is produced in porcine adrenal glands and testes, and may regulate cardiovascular functions through eNOS expression.

Notch and TNF-α signaling promote cytoplasmic accumulation of OLFM4 in intestinal epithelium cells and exhibit a cell protective role in the inflamed mucosa of IBD patients.

Notch signaling is activated in the intestinal epithelial cells (IECs) of patients with inflammatory bowel disease (IBD), and contributes to mucosal regeneration. Our previous study indicated that TNF-α and Notch signaling may synergistically promote the expression of the intestinal stem cell (ISC) marker OLFM4 in human IECs. In the present study, we investigated the gene regulation and function of OLFM4 in human IEC lines. We confirmed that TNF-α and Notch synergistically upregulate the mRNA expression of OLFM4. Luciferase reporter assay showed that OLFM4 transcription is regulated by the synergy of TNF-α and Notch. At the protein level, synergy between TNF-α and Notch promoted cytoplasmic accumulation of OLFM4, which has potential anti-apoptotic properties in human IECs. Analysis of patient-derived tissues and organoids consistently showed cytoplasmic accumulation of OLFM4 in response to NF-κB and Notch activation. Cytoplasmic accumulation of OLFM4 in human IECs is tightly regulated by Notch and TNF-α in synergy. Such cytoplasmic accumulation of OLFM4 may have a cell-protective role in the inflamed mucosa of patients with IBD.

Functional analysis of isoflavones using patient-derived human colonic organoids.

Inflammatory bowel disease (IBD) comprises two major subtypes, ulcerative colitis (UC) and Crohn's disease, which are multifactorial diseases that may develop due to genetic susceptibility, dysbiosis, or environmental factors. Environmental triggers of IBD include food-borne factors, and a previous nationwide survey in Japan identified pre-illness consumption of isoflavones as a risk factor for UC. However, the precise mechanisms involved in the detrimental effects of isoflavones on the intestinal mucosa remain unclear. The present study employed human colonic organoids (hCOs) to investigate the functional effect of two representative isoflavones, genistein and daidzein, on human colonic epithelial cells. The addition of genistein to organoid reformation assays significantly decreased the number and size of reformed hCOs compared with control and daidzein treatment, indicating an inhibitory effect of genistein on colonic cell/progenitor cell function. Evaluation of the phosphorylation status of 49 different receptor tyrosine kinases showed that genistein selectively inhibited phosphorylation of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR). We established a two-dimensional wound-repair model using hCOs and showed that genistein significantly delayed the overall wound-repair response. Our results collectively show that genistein may exert its detrimental effects on the intestinal mucosa via negative regulation of stem/progenitor cell function, possibly leading to sustained mucosal injury and the development of UC.

The diagnostic and prognostic value of mature and total adrenomedullin for sepsis: a prospective observational study.

INTRODUCTION: Information about biologically active adrenomedullin (mature AM), a potential new biomarker for sepsis and septic shock, is limited. Here, we investigated the value of mature AM for diagnosis and outcome prediction in sepsis. MATERIAL AND METHODS: Patients admitted to the intensive care unit (ICU) were retrospectively cate-gorised into non-sepsis or sepsis groups, according to the Sepsis-3 definitions. Plasma levels of mature and total (the sum of the levels of intermediate and mature forms) AM were measured, and their usefulness was compared with that of other sepsis biomarkers, such as procalcitonin and presepsin. RESULTS: Of the 98 patients analysed, 42 were assigned to the non-sepsis and 56 to the sepsis group. Mature and total AM levels on admission were significantly higher in patients with than in those without sepsis. The areas under the receiver operating characteristic curves (AUCs) of mature and total AM for diagnosing sepsis were 0.85 and 0.88, whereas those of procalcitonin and presepsin were 0.83 and 0.68, respectively. AUCs of mature and total AM for predicting 28-day mortality in patients with sepsis became significant on day 3 after admission. A good correlation between the AM forms was found, indicating that changes in their plasma levels may directly reflect each other. CONCLUSIONS: Because mature and total AM levels increased significantly in patients with sepsis on admission, both forms may be used as reliable and early biomarkers for diagnosing sepsis according to the Sepsis-3 definitions. However, prediction of 28-day mortality in such patients would require several days of ICU stay.

Development of a novel AlphaLISA ImmunoAssay for Big angiotensin-25.

We previously described the discovery of Big angiotensin-25 (Bang-25), an angiotensin-related peptide isolated from human urine. Bang-25 consists of the first 25 amino acids of the N-terminus of angiotensinogen (Aogen), with N-linked glycosylation on the 14th amino acid and a cysteine conjugated to the 18th amino acid. Bang-25 is rapidly converted into angiotensin II (Ang II) by chymase. Because Bang-25 is widely distributed in human tissues, including islet cells in the pancreas and podocytes in the kidney, we hypothesized that it may participate in the Ang II production system in these tissues. To test this hypothesis, we developed a specific assay for Bang-25 and used it to examine the urinary concentrations of Bang-25 in patients with diabetes mellitus (DM). The assay used the Amplified Luminescent Proximity Homogeneous Assay (Alpha)-based ELISA method (AlphaLISA) of PerkinElmer Japan and included antibodies specific to the N-terminus of Ang II and the C-terminus of Bang-25. The AlphaLISA ImmunoAssay specifically recognized Bang-25 and had no cross-reactivity with Aogen or Ang I. Bang-25 was detected in healthy volunteers' urine samples but not in their plasma samples. In patients with DM, the urinary Bang-25 concentration was significantly higher than in healthy volunteers. Moreover, the results indicated that the Bang-25 concentration in the urine may offer a different perspective on disease status from that provided by urinary albumin. This assay could provide a useful tool for determining urinary Bang-25, which may prove an important biomarker for diabetic kidney disease.

Activation of Calcitonin Gene-Related Peptide and Adrenomedullin Receptors by PEGylated Adrenomedullin.

Adrenomedullin (AM) improves colitis in animal models and patients with inflammatory bowel disease. We have developed a PEGylated AM derivative (PEG-AM) for clinical application because AM has a short half-life in the blood. However, modification by addition of polyethylene glycol (PEG) may compromise the function of the original peptide. In this paper, we examined the time course of cAMP accumulation induced by 5 and 60 kDa PEG-AM and compared the activation of calcitonin gene-related peptide (CGRP), AM1 and AM2 receptors by AM, 5 and 60 kDa PEG-AM. We also evaluated the effects of antagonists on the action of 5 and 60 kDa PEG-AM. PEG-AM stimulated cAMP production induced by these receptors; the increase in cAMP levels resulting from application of PEG-AM peaked at 15 min. Moreover, PEG-AM activity was antagonized by CGRP (8-37) or AM (22-52) (antagonists of CGRP and AM receptors, respectively) and the maximal response was not suppressed. These findings indicate that the effects of PEG-AM are similar to those of native AM.

Thrombin rapidly digests adrenomedullin: Synthesis of adrenomedullin analogs resistant to thrombin.

Human adrenomedullin (AM) functions as a circulating hormone and as a local paracrine mediator with multiple biological activities. We investigated the metabolism of AM by examining its fragmentation in human serum. Adrenomedullin was rapidly cleaved in human serum, but was relatively stable in plasma. We showed that AM was rapidly digested by thrombin in serum, with AM(13-44) as the main product. On the basis of these data, we prepared AM analogs in which Arg-44 was replaced by Ala, Lys, and D-Arg, respectively. These analogs were resistant to thrombin and showed comparable biological activity to native AM. Furthermore, the bioavailabilities of these peptides were improved after subcutaneous administration in rats. These AM analogs may be promising drug candidates for clinical applications.

Atrial angiotensin-(1-12)/chymase expression data in patient of heart diseases.

The data presented here are related to the research article entitled "Differential expression of the angiotensin-(1-12) [Ang-(1-12)]/chymase axis in human atrial tissue [1]. We have showed that chymase gene transcripts, chymase activity, and immunoreactive- Ang-(1-12) expression levels were higher in left compared to right atrial tissue, irrespective of cardiac disease. This article presents the echocardiographic characteristics of 111 patients undergoing heart surgery for the correction of valvular heart disease, resistant atrial fibrillation or ischemic heart disease. Left atrial chymase mRNA expression and activity, and left atrial Ang-(1-12) levels were compared between patients with stroke vs. non-stroke, congestive heart failure vs. non-heart failure, and in cardiac surgery patients who had a history of postoperative atrial fibrillation vs. non-atrial fibrillation.

Differential Expression of the Angiotensin-(1-12)/Chymase Axis in Human Atrial Tissue.

BACKGROUND: Heart chymase rather than angiotensin (Ang)-converting enzyme has higher specificity for Ang I conversion into Ang II in humans. A new pathway for direct cardiac Ang II generation has been revealed through the demonstration that Ang-(1-12) is cleaved by chymase to generate Ang II directly. Herein, we address whether Ang-(1-12), chymase messenger RNA (mRNA), and activity levels can be differentiated in human atrial tissue from normal and diseased hearts and if these measures associate with various pathologic heart conditions. MATERIALS AND METHODS: Atrial appendages were collected from 11 nonfailing donor hearts and 111 patients undergoing heart surgery for the correction of valvular heart disease, resistant atrial fibrillation, or ischemic heart disease. Chymase mRNA was analyzed by real-time polymerase chain reaction and enzymatic activity by high-performance liquid chromatography using Ang-(1-12) as the substrate. Ang-(1-12) levels were determined by immunohistochemical staining. RESULTS: Chymase gene transcripts, chymase activity, and immunoreactive Ang-(1-12) expression levels were higher in left atrial tissue compared with right atrial tissue, irrespective of cardiac disease. In addition, left atrial chymase mRNA expression was significantly higher in stroke versus nonstroke patients and in cardiac surgery patients who had a history of postoperative atrial fibrillation versus nonatrial fibrillation. Correlation analysis showed that left atrial chymase mRNA was positively related to left atrial enlargement, as determined by echocardiography. CONCLUSIONS: As Ang-(1-12) expression and chymase gene transcripts and enzymatic activity levels were positively linked to left atrial size in patients with left ventricular heart disease, an important alternate Ang II forming pathway, via Ang-(1-12) and chymase, in maladaptive atrial and ventricular remodeling in humans is uncovered.

TGF-ß promotes fetal gene expression and cell migration velocity in a wound repair model of untransformed intestinal epithelial cells.

The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-ß. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1, Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-ß. Surprisingly, addition of TGF-ß induced a set of genes, including Slc28a2, Tubb2a, and Cpe, that were expressed preferentially in fetal IECs. Moreover, TGF-ß significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs.

Organoid-based regenerative medicine for inflammatory bowel disease.

Inflammatory bowel disease (IBD) consists of two major idiopathic gastrointestinal diseases: ulcerative colitis and Crohn's disease. Although a significant advance has been achieved in the treatment of IBD, there remains a particular population of patients that are refractory to the conventional treatments, including the biologic agents. Studies have revealed the importance of "mucosal healing" in improving the prognosis of those difficult-to-treat patients, which indicates the proper and complete regeneration of the damaged intestinal tissue. In this regard, organoid-based regenerative medicine may have the potential to dramatically promote the achievement of mucosal healing in refractory IBD patients, and thereby improve their long-term prognosis as well. So far, studies have shown that hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) may have some beneficial effect on IBD patients through their transplantation or transfusion. Recent advance in stem cell biology has added intestinal stem cells (ISCs) as a new player in this field. It has been shown that ISCs can be grown in vitro as organoids and that those ex-vivo cultured organoids can be employed as donor cells for transplantation studies. Further studies using mice colitis models have shown that ex-vivo cultured organoids can engraft onto the colitic ulcers and reconstruct the crypt-villus structures. Such transplantation of organoids may not only facilitate the regeneration of the refractory ulcers that may persist in IBD patients but may also reduce the risk of developing colitis-associated cancers. Endoscopy-assisted transplantation of organoids may, therefore, become one of the alternative therapies for refractory IBD patients.