• BMB Reports
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• v.56 no.1
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• pp.15-23
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• 2023

After birth, animals are colonized by a diverse community of microorganisms. The digestive tract is known to contain the largest number of microbiome in the body. With emergence of the gut-brain axis, the importance of gut microbiome and its metabolites in host health has been extensively studied in recent years. The establishment of organoid culture systems has contributed to studying intestinal pathophysiology by replacing current limited models. Owing to their architectural and functional complexity similar to a real organ, co-culture of intestinal organoids with gut microbiome can provide mechanistic insights into the detrimental role of pathobiont and the homeostatic function of commensal symbiont. Here organoid-based bacterial co-culture techniques for modeling host-microbe interactions are reviewed. This review also summarizes representative studies that explore impact of enteric microorganisms on intestinal organoids to provide a better understanding of host-microbe interaction in the context of homeostasis and disease.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.136-143
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• 2022

Recent progress has been made to establish intestinal organoids for an in vitro model as a potential alternative to an in vivo system in animals. We previously reported a reliable method for the isolation of intestinal crypts from the small intestine and robust three-dimensional (3D) expansion of intestinal organoids (basal-out) in adult bovines. The present study aimed to establish next-generation intestinal organoids for practical applications in disease modeling-based host-pathogen interactions and feed efficiency measurements. In this study, we developed a rapid and convenient method for the efficient generation of intestinal organoids through the modulation of the Wnt signaling pathway and continuous apical-out intestinal organoids. Remarkably, the intestinal epithelium only takes 3-4 days to undergo CHIR (1 µM) treatment as a Wnt activator, which is much shorter than that required for spontaneous differentiation (7 days). Subsequently, we successfully established an apical-out bovine intestinal organoid culture system through suspension culture without Matrigel matrix, indicating an apical-out membrane on the surface. Collectively, these results demonstrate the efficient generation and next-generation of bovine intestinal organoids and will facilitate their potential use for various purposes, such as disease modeling, in the field of animal biotechnology.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.2765-2769
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• 2013

Background: A close association between patterns identified by magnifying narrow-band imaging (M-NBI) and histological type has been described. M-NBI patterns were also recently reported to be related to the mucin phenotype; however, detials remain unclear. Materials and Methods: We investigated the cellular differentiation of gastric cancer lesions, along with their mucosal distribution observed by M-NBI. Ninety-seven depressed-type early gastric cancer lesions (74 differentiated and 23 undifferentiated adenocarcinomas) were visualized by M-NBI. Findings were divided into 4 patterns based on abnormal microvascular architecture: a chain loop pattern (CLP), a fine network pattern (FNP), a corkscrew pattern (CSP), and an unclassified pattern. Mucin phenotypes were judged as gastric (G-type), intestinal (I-type), mixed gastric and intestinal (M-type), and null (N-type) based on 4 markers (MAC5AC, MUC6, MUC2, and CD10). The relationship of each pattern of microvascular architecture with organoid differentiation indicated by cancer cell differentiation and its distribution in each histological type of early gastric cancer was investigated. Results: All CLP and FNP lesions were differentiated. The cancer cell distribution showed organoid differentiation in 84.2% (16/19) and 61.1% (22/36) of the two types of lesions, respectively, and there was a significant difference from the unclassified pattern with organoid differentiation (p<0.001). Almost all (94.7%; 18/19) CSP lesions were undifferentiated, and organoid differentiation was observed in 72.2% (13/18). There was a significant difference from the unclassified pattern with organoid differentiation (p<0.05). Conclusions: Cellular differentiation and distribution are associated with microvascular architecture observed by M-NBI.

• Molecules and Cells
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• v.41 no.4
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• pp.290-300
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• 2018

Using an in vitro model of intestinal organoids derived from intestinal crypts, we examined effects of indole-3-carbinol (I3C), a phytochemical that has anticancer and aryl hydrocarbon receptor (AhR)-activating abilities and thus is sold as a dietary supplement, on the development of intestinal organoids and investigated the underlying mechanisms. I3C inhibited the in vitro development of mouse intestinal organoids. Addition of ${\alpha}$-naphthoflavone, an AhR antagonist or AhR siRNA transfection, suppressed I3C function, suggesting that I3C-mediated interference with organoid development is AhR-dependent. I3C increased the expression of Muc2 and lysozyme, lineage-specific genes for goblet cells and Paneth cells, respectively, but inhibits the expression of IAP, a marker gene for enterocytes. In the intestines of mice treated with I3C, the number of goblet cells was reduced, but the number of Paneth cells and the depth and length of crypts and villi were not changed. I3C increased the level of active nonphosphorylated ${\beta}$-catenin, but suppressed the Notch signal. As a result, expression of Hes1, a Notch target gene and a transcriptional repressor that plays a key role in enterocyte differentiation, was reduced, whereas expression of Math1, involved in the differentiation of secretory lineages, was increased. These results provide direct evidence for the role of AhR in the regulation of the development of intestinal stem cells and indicate that such regulation is likely mediated by regulation of Wnt and Notch signals.

• Journal of Animal Reproduction and Biotechnology
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• v.39 no.1
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• pp.2-11
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• 2024

Background: The small intestine plays a crucial role in animals in maintaining homeostasis as well as a series of physiological events such as nutrient uptake and immune function to improve productivity. Research on intestinal organoids has recently garnered interest, aiming to study various functions of the intestinal epithelium as a potential alternative to an in vivo system. These technologies have created new possibilities and opportunities for substituting animals for testing with an in vitro model. Methods: Here, we report the establishment and characterisation of intestinal organoids derived from jejunum tissues of adult pigs. Intestinal crypts, including intestinal stem cells from the jejunum tissue of adult pigs (10 months old), were sequentially isolated and cultivated over several passages without losing their proliferation and differentiation using the scaffold-based and three-dimensional method, which indicated the recapitulating capacity. Results: Porcine jejunum-derived intestinal organoids showed the specific expression of several genes related to intestinal stem cells and the epithelium. Furthermore, they showed high permeability when exposed to FITC-dextran 4 kDa, representing a barrier function similar to that of in vivo tissues. Collectively, these results demonstrate the efficient cultivation and characteristics of porcine jejunum-derived intestinal organoids. Conclusions: In this study, using a 3D culture system, we successfully established porcine jejunum-derived intestinal organoids. They show potential for various applications, such as for nutrient absorption as an in vitro model of the intestinal epithelium fused with organ-on-a-chip technology to improve productivity in animal biotechnology in future studies.

• Journal of Veterinary Science
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• v.24 no.4
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• pp.53.1-53.12
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• 2023

Background: Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied. Objectives: This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection. Methods: PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity. Results: The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner. Conclusions: We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

• Journal of Animal Science and Technology
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• v.64 no.6
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• pp.1105-1116
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• 2022

Recently, we reported the robust in vitro three-dimensional (3D) expansion of intestinal organoids derived from adult bovine (> 24 months) samples. The present study aimed to establish an in vitro 3D system for the cultivation of intestinal organoids derived from growing cattle (12 months old) for practical use as a potential alternative to in vivo systems for various purposes. However, very few studies on the functional characterization and 3D expansion of adult stem cells from livestock species compared to those from other species are available. In this study, intestinal crypts, including intestinal stem cells, from the small intestines (ileum and jejunum) of growing cattle were isolated and long-term 3D cultures were successfully established using a scaffold-based method. Furthermore, we generated an apical-out intestinal organoid derived from growing cattle. Interestingly, intestinal organoids derived from the ileum, but not the jejunum, could be expanded without losing the ability to recapitulate crypts, and these organoids specifically expressed several specific markers of intestinal stem cells and the intestinal epithelium. Furthermore, these organoids exhibited key functionality with regard to high permeability for compounds up to 4 kDa in size (e.g., fluorescein isothiocyanate [FITC]-dextran), indicating that apical-out intestinal organoids are better than other models. Collectively, these results indicate the establishment of growing cattle-derived intestinal organoids and subsequent generation of apical-out intestinal organoids. These organoids may be valuable tools and potential alternatives to in vivo systems for examining host-pathogen interactions involving epithelial cells, such as enteric virus infection and nutrient absorption, and may be used for various purposes.