Resveratrol (RSV) is a natural polyphenol that has a beneficial effect on health, and resveratrol-induced autophagy has been suggested to be a key process in mediating many beneficial effects of ...resveratrol, such as reduction of inflammation and induction of cancer cell death. Although various resveratrol targets have been suggested, the molecule that mediates resveratrol-induced autophagy remains unknown. Here, we demonstrate that resveratrol induces autophagy by directly inhibiting the mTOR-ULK1 pathway. We found that inhibition of mTOR activity and presence of ULK1 are required for autophagy induction by resveratrol. In line with this mTOR dependency, we found that resveratrol suppresses the viability of MCF7 cells but not of SW620 cells, which are mTOR inhibitor sensitive and insensitive cancer cells, respectively. We also found that resveratrol-induced cancer cell suppression occurred ULK1 dependently. For the mechanism of action of resveratrol on mTOR inhibition, we demonstrate that resveratrol directly inhibits mTOR. We found that resveratrol inhibits mTOR by docking onto the ATP-binding pocket of mTOR (i.e., it competes with ATP). We propose mTOR as a novel direct target of resveratrol, and inhibition of mTOR is necessary for autophagy induction.
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.
Three-dimensional human intestinal organoids (hIO) are widely used as a platform for biological and biomedical research. However, reproducibility and challenges for large-scale expansion limit their ...applicability. Here, we establish a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC
) within hIO derived from human pluripotent stem cells. The intrinsic self-organisation property of ISC
, combined with air-liquid interface culture in a minimally defined medium, forces ISC
to differentiate into the intestinal epithelium with cellular diversity, villus-like structure, and barrier integrity. Notably, ISC
is an ideal cell source for gene editing to study ISC biology and transplantation for intestinal diseases. We demonstrate the intestinal epithelium differentiated from ISC
as a model system to study severe acute respiratory syndrome coronavirus 2 viral infection. ISC
culture technology provides a biological tool for use in regenerative medicine and disease modelling.
The morphology and structure of the intestinal epithelium are rearranged dynamically during development, tissue regeneration, and disease progression. The most important characteristic of intestinal ...epithelial morphogenesis is the repetitive compartmentalized structures of crypt-villus units, which are crucial for maintaining intestinal homeostasis and functions. Abnormal structures are known to be closely associated with disease development and progression. Therefore, understanding how intestinal crypt-villus structures are formed and grown is essential for elucidating the physiological and pathophysiological roles of the intestinal epithelium. However, a critical knowledge gap in understanding the compartmentalization of the crypt-villus axis remains when using animal models, due to obvious inter-species differences and difficulty in real-time monitoring. Recently, emerging technologies such as organoid culture, lineage tracing, and single cell sequencing have enabled the assessment of the intrinsic mechanisms of intestinal epithelial morphogenesis. In this review, we discuss the latest research on the regulatory factors and signaling pathways that play a central role in the formation, maintenance, and regeneration of crypt-villus structures in the intestinal epithelium. Furthermore, we discuss how these factors and pathways play a role in development, tissue regeneration, and disease. We further explore how the current technology of three-dimensional intestinal organoids has contributed to the understanding of crypt-villus compartmentalization, highlighting new findings related to the self-organizing-process-driven initiation and propagation of crypt-villus structures. We also discuss intestinal diseases featuring abnormalities of the crypt-villus structure to provide insights for the development of novel therapeutic strategies targeting intestinal morphogenesis and crypt-villus formation.
Several phase 1/2 clinical trials showed that low-dose interleukin-2 (IL-2) treatment is a safe and effective strategy for the treatment of chronic graft-versus-host disease, hepatitis C ...virus-induced vasculitis, and type 1 diabetes. Ulcerative colitis (UC) is a chronic inflammatory condition of the colon that lacks satisfactory treatment. In this study, we aimed to determine the effects of low-dose IL-2 as a therapeutic for UC on dextran sulfate sodium (DSS)-induced colitis.
: Mice with DSS-induced colitis were intraperitoneally injected with low-dose IL-2. Survival, body weight, disease activity index, colon length, histopathological score, myeloperoxidase activity and inflammatory cytokine levels as well as intestinal barrier integrity were examined. Differential gene expression after low-dose IL-2 treatment was analyzed by RNA-sequencing.
: Low-dose IL-2 significantly improved the symptoms of DSS-induced colitis in mice and attenuated pro-inflammatory cytokine production and immune cell infiltration. The most effective dose range of IL-2 was 16K-32K IU/day. Importantly, low-dose IL-2 was effective in ameliorating the disruption of epithelial barrier integrity in DSS-induced colitis tissues by restoring tight junction proteins and mucin production and suppressing apoptosis. The colon tissue of DSS-induced mice exposed to low-dose IL-2 mimic gene expression patterns in the colons of control mice. Furthermore, we identified the crucial role of the PI3K-AKT pathway in exerting the therapeutic effect of low-dose IL-2.
: The results of our study suggest that low-dose IL-2 has therapeutic effects on DSS-induced colitis and potential clinical value in treating UC.
Little is known about the modulatory capacity of the microbiota in early intestinal development. We examined various intestinal models that respond to gut microbial metabolites based on human ...pluripotent stem cell-derived human intestinal organoids (hIOs): physiologically relevant in vitro fetal-like intestine, intestinal stem cell, and intestinal disease models. We found that a newly isolated Limosilactobacillus reuteri strain DS0384 accelerated maturation of the fetal intestine using 3D hIO with immature fetal characteristics. Comparative metabolomic profiling analysis revealed that the secreted metabolite N-carbamyl glutamic acid (NCG) is involved in the beneficial effect of DS0384 cell-free supernatants on the intestinal maturation of hIOs. Experiments in an intestinal stem cell spheroid model and hIO-based intestinal inflamed model revealed that the cell-free supernatant from DS0384 comprising NCG promoted intestinal stem cell proliferation and was important for intestinal protection against cytokine-induced intestinal epithelial injury. The probiotic properties of DS0384 were also evaluated, including acid and bile tolerance and ability to adhere to human intestinal cells. Seven-day oral administration of DS0384 and cell-free supernatant promoted the intestinal development of newborn mice. Moreover, NCG exerted a protective effect on experimental colitis in mice. These results suggest that DS0384 is a useful agent for probiotic applications and therapeutic treatment for disorders of early gut development and for preventing intestinal barrier dysfunction.
Lysosomal localization of mammalian target of rapamycin complex 1 (mTORC1) is a critical step for activation of the molecule. Rag GTPases are essential for this translocation. Here, we demonstrate ...that Nudix-type motif 2 (NUDT2) is a novel positive regulator of mTORC1 activation. Activation of mTORC1 is impaired in NUDT2-silenced cells. Mechanistically, NUDT2 binds to Rag GTPase and controls mTORC1 translocation to the lysosomal membrane. Furthermore, NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells, as NUDT2-silenced cells arrest in G0/G1 phases. Taken together, these results show that NUDT2 is a novel complex formation enhancing factor regulating mTORC1-Rag GTPase signaling that is crucial for cell growth control.
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•NUDT2 is identified as a novel regulator of Rag GTPase-mTORC1 signaling.•NUDT2 was associated with recruitment of mTORC1 to the lysosomal membrane.•The physical interaction between NUDT2 and Rag GTPase heterodimers is required for the lysosomal localization of mTORC1.•NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells.
Advanced technologies are required for generating human intestinal epithelial cells (hIECs) harboring cellular diversity and functionalities to predict oral drug absorption in humans and study normal ...intestinal epithelial physiology. We developed a reproducible two-step protocol to induce human pluripotent stem cells to differentiate into highly expandable hIEC progenitors and a functional hIEC monolayer exhibiting intestinal molecular features, cell type diversity, and high activities of intestinal transporters and metabolic enzymes such as cytochrome P450 3A4 (CYP3A4). Functional hIECs are more suitable for predicting compounds metabolized by CYP3A4 and absorbed in the intestine than Caco-2 cells. This system is a step toward the transition from three-dimensional (3D) intestinal organoids to 2D hIEC monolayers without compromising cellular diversity and function. A physiologically relevant hIEC model offers a novel platform for creating patient-specific assays and support translational applications, thereby bridging the gap between 3D and 2D culture models of the intestine.
Human intestinal organoids (hIOs), which resemble the human intestine structurally and physiologically, have emerged as a new modality for the study of the molecular and cellular biology of the ...intestine in vitro. We recently developed an in vitro maturation technique for generating functional hIOs from human pluripotent stem cells (hPSCs). Here, we investigated the function of STAT3 for inducing in vitro maturation of hIOs. This was accompanied by the tyrosine phosphorylation of STAT3, whereas treatment with pharmacological inhibitors of STAT3 suppressed the phosphorylation of STAT3 and the expression of intestinal maturation markers. We generated and characterized STAT3 knockout (KO) human embryonic stem cell (hESC) lines using CRISPR/Cas9-mediated gene editing. We found that STAT3 KO does not affect the differentiation of hESCs into hIOs but rather affects the in vitro maturation of hIOs. STAT3 KO hIOs displayed immature morphologies with decreased size and reduced budding in hIOs even after in vitro maturation. STAT3 KO hIOs showed markedly different profiles from hIOs matured in vitro and human small intestine. Additionally, STAT3 KO hIOs failed to maintain upon in vivo transplantation. This study reveals a core signaling pathway consisting of STAT3 controlling the in vitro maturation of hIOs derived from hPSCs.
The gastrointestinal tract is the most common exposure route of xenobiotics, and intestinal toxicity can result in systemic toxicity in most cases. It is important to develop intestinal toxicity ...assays mimicking the human system; thus, stem cells are rapidly being developed as new paradigms of toxicity assessment. In this study, we established human embryonic stem cell (hESC)-derived enterocyte-like cells (ELCs) and compared them to existing
in vivo
and
in vitro
models. We found that hESC-ELCs and the
in vivo
model showed transcriptomically similar expression patterns of a total of 10,020 genes than the commercialized cell lines. Besides, we treated the hESC-ELCs,
in vivo
rats, Caco-2 cells, and Hutu-80 cells with quarter log units of lethal dose 50 or lethal concentration 50 of eight drugs—chloramphenicol, cycloheximide, cytarabine, diclofenac, fluorouracil, indomethacin, methotrexate, and oxytetracycline—and then subsequently analyzed the biomolecular markers and morphological changes. While the four models showed similar tendencies in general toxicological reaction, hESC-ELCs showed a stronger correlation with the
in vivo
model than the immortalized cell lines. These results indicate that hESC-ELCs can serve as a next-generation intestinal toxicity model.
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