• International Journal of Stem Cells
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• 제17권1호
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• pp.51-58
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• 2024

With the activity of intestinal stem cells and continuous turnover, the gut epithelium is one of the most dynamic tissues in animals. Due to its simple yet conserved tissue structure and enteric cell composition as well as advanced genetic and histologic techniques, Drosophila serves as a valuable model system for investigating the regulation of intestinal stem cells. The Drosophila gut epithelium is in constant contact with indigenous microbiota and encounters externally introduced "non-self" substances, including foodborne pathogens. Therefore, in addition to its role in digestion and nutrient absorption, another essential function of the gut epithelium is to control the expansion of microbes while maintaining its structural integrity, necessitating a tissue turnover process involving intestinal stem cell activity. As a result, the microbiome and pathogens serve as important factors in regulating intestinal tissue turnover. In this manuscript, I discuss crucial discoveries revealing the interaction between gut microbes and the host's innate immune system, closely associated with the regulation of intestinal stem cell proliferation and differentiation, ultimately contributing to epithelial homeostasis.

• 생명과학회지
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• 제22권5호
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• pp.621-626
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• 2012

초파리는 발생과 질병연구를 위한 모델 곤충으로 널리 이용되어 왔다. 본 연구에서도 초파리를 모델곤충으로 한 장질환 연구의 일환으로 다양한 병원균 감염 및 장질환 유발시 어떻게 장줄기세포가 작용하는지를 이해하기 위해 초파리 장세포의 일차배양 방법을 확립하였다. 초파리 성충으로부터 장을 해부하고 다양한 효소를 처리하여 장세포를 분리한 후 배양하였다. 배양세포의 생존여부는 현미경 검경 및 MTS assay에 의해 확인한 결과 배양 후 9일째 최대 증식되었고 14일까지 생존함을 확인할 수 있었다. 또한 장줄기세포 및 장내분비세포의 존재도 immunostaining에 의해 확인하였다. 따라서 본 연구에서 구축된 초파리 일차배양 장세포는 다양한 유전자에 의한 장줄기 세포 조절연구뿐만 아니라 장에서 발생하는 다양한 질병을 연구하는 도구로 매우 유용할 것으로 추측된다.

• Journal of Animal Science and Technology
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• 제64권6호
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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.

• 한국동물생명공학회지
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• 제39권1호
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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.

• 대한수의학회지
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• 제56권2호
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• pp.57-66
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• 2016

The gut epithelial barrier, which is composed of the mucosal layer and the intestinal epithelium, has multiple defense mechanisms and interconnected regulatory mechanisms against enteric microbial pathogens. However, many bacterial pathogens have highly evolved infectious stratagems that manipulate mucin production, epithelial cell-cell junctions, cell death, and cell turnover to promote their replication and pathogenicity in the gut epithelial barrier. In this review, we focus on current knowledge about how bacterial pathogens regulate mucin levels to circumvent the epithelial mucus barrier and target cell-cell junctions to invade deeper tissues and increase their colonization. We also describe how bacterial pathogens manipulate various modes of epithelial cell death to facilitate bacterial dissemination and virulence effects. Finally, we discuss recent investigating how bacterial pathogens regulate epithelial cell turnover and intestinal stem cell populations to modulate intestinal epithelium homeostasis.

• Molecules and Cells
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• 제39권4호
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• pp.358-366
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• 2016

The digestive system is gaining interest as a major regulator of various functions including immune defense, nutrient accumulation, and regulation of feeding behavior, aside from its conventional function as a digestive organ. The Drosophila midgut epithelium is completely renewed every 1-2 weeks due to differentiation of pluripotent intestinal stem cells in the midgut. Intestinal stem cells constantly divide and differentiate into enterocytes that secrete digestive enzymes and absorb nutrients, or enteroendocrine cells that secrete regulatory peptides. Regulatory peptides have important roles in development and metabolism, but study has mainly focused on expression and functions in the nervous system, and not much is known about the roles in endocrine functions of enteroendocrine cells. We systemically examined the expression of 45 regulatory peptide genes in the Drosophila midgut, and verified that at least 10 genes are expressed in the midgut enteroendocrine cells through RT-PCR, in situ hybridization, antisera, and 25 regulatory peptide-GAL transgenes. The Drosophila midgut is highly compartmentalized, and individual peptides in enteroendocrine cells were observed to express in specific regions of the midgut. We also confirmed that some peptides expressed in the same region of the midgut are expressed in mutually exclusive enteroendocrine cells. These results indicate that the midgut enteroendocrine cells are functionally differentiated into different subgroups. Through this study, we have established a basis to study regulatory peptide functions in enteroendocrine cells as well as the complex organization of enteroendocrine cells in the Drosophila midgut.

• Journal of Microbiology and Biotechnology
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• 제33권10호
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• pp.1309-1316
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• 2023

To exert their beneficial effects, it is essential for the commensal bacteria of probiotic supplements to be sufficiently protected as they pass through the low pH environment of the stomach, and effectively colonize the intestinal epithelium downstream. Here, we investigated the effect of a multilayer coating containing red ginseng dietary fiber, on the acid tolerance, and the adhesion and proliferation capacities of three Lactobacillus strains (Limosilactobacillus reuteri KGC1901, Lacticaseibacillus casei KGC1201, Limosilactobacillus fermentum KGC1601) isolated from Panax ginseng, using HT-29 cells, mucin-coated plates, and human pluripotent stem cell-derived intestinal epithelial cells as in vitro models of human gut physiology. We observed that the multilayer-coated strains displayed improved survival rates after passage through gastric juice, as well as high adhesion and proliferation capacities within the various gut epithelial systems tested, compared to their uncoated counterparts. Our findings demonstrated that the multilayer coat effectively protected commensal microbiota and led to improved adhesion and colonization of intestinal epithelial cells, and consequently to higher probiotic efficacy.

• Molecules and Cells
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• 제41권4호
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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.

• Parasites, Hosts and Diseases
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• 제59권6호
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• pp.573-583
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• 2021

Toxoplasma gondii, an intracellular protozoan parasite that infects one-third of the world's population, has been reported to hijack host cell apoptotic machinery and promote either an anti- or proapoptotic program depending on the parasite virulence and load and the host cell type. However, little is known about the regulation of human FHs 74 small intestinal epithelial cell viability in response to T. gondii infection. Here we show that T. gondii RH strain tachyzoite infection or ESP treatment of FHs 74 Int cells induced apoptosis, mitochondrial dysfunction and ER stress in host cells. Pretreatment with 4-PBA inhibited the expression or activation of key molecules involved in ER stress. In addition, both T. gondii and ESP challenge-induced mitochondrial dysfunction and cell death were dramatically suppressed in 4-PBA pretreated cells. Our study indicates that T. gondii infection induced ER stress in FHs 74 Int cells, which induced mitochondrial dysfunction followed by apoptosis. This may constitute a potential molecular mechanism responsible for the foodborne parasitic disease caused by T. gondii.

• Molecules and Cells
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• 제46권8호
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• pp.476-485
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• 2023

Gastric cancer stem-like cells (GCSCs) possess stem cell properties, such as self-renewal and tumorigenicity, which are known to induce high chemoresistance and metastasis. These characteristics of GCSCs are further enhanced by autophagy, worsening the prognosis of patients. Currently, the mechanisms involved in the induction of stemness in GCSCs during autophagy remain unclear. In this study, we compared the cellular responses of GCSCs with those of gastric cancer intestinal cells (GCICs) whose stemness is not induced by autophagy. In response to glucose starvation, the levels of β-catenin and stemness-related genes were upregulated in GCSCs, while the levels of β-catenin declined in GCICs. The pattern of deubiquitinase ubiquitin C-terminal hydrolase-L3 (UCH-L3) expression in GCSCs and GCICs was similar to that of β-catenin expression depending on glucose deprivation. We also observed that inhibition of UCH-L3 activity reduced β-catenin protein levels. The interaction between UCH-L3 and β-catenin proteins was confirmed, and it reduced the ubiquitination of β-catenin. Our results suggest that UCH-L3 induces the stabilization of β-catenin, which is required to promote stemness during autophagy activation. Also, UCH-L3 expression was regulated by c-Fos, and the levels of c-Fos increased in response to autophagy activation. In summary, our findings suggest that the inhibition of UCH-L3 during nutrient deprivation could suppress stress resistance of GCSCs and increase the survival rates of gastric cancer patients.