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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 (ISC3D-hIO) within hIO derived from human pluripotent stem cells. The intrinsic self-organisation property of ISC3D-hIO, combined with air-liquid interface culture in a minimally defined medium, forces ISC3D-hIO to differentiate into the intestinal epithelium with cellular diversity, villus-like structure, and barrier integrity. Notably, ISC3D-hIO is an ideal cell source for gene editing to study ISC biology and transplantation for intestinal diseases. We demonstrate the intestinal epithelium differentiated from ISC3D-hIO as a model system to study severe acute respiratory syndrome coronavirus 2 viral infection. ISC3D-hIO culture technology provides a biological tool for use in regenerative medicine and disease modelling.
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Intestinos , Células Madre Pluripotentes , Humanos , Reproducibilidad de los Resultados , Mucosa Intestinal , Organoides , Diferenciación CelularRESUMEN
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.
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Lactobacillus , Probióticos , Humanos , Probióticos/farmacología , Mucosa Intestinal/microbiología , Células Epiteliales/microbiología , Proliferación CelularRESUMEN
Background: A growing body of evidence suggests that particulate matter (PM10) enters the gastrointestinal (GI) tract directly, causing the GI epithelial cells to function less efficiently, leading to inflammation and an imbalance in the gut microbiome. PM10 may, however, act as an exacerbation factor in patients with inflamed intestinal epithelium, which is associated with inflammatory bowel disease. Objective: The purpose of this study was to dissect the pathology mechanism of PM10 exposure in inflamed intestines. Methods: In this study, we established chronically inflamed intestinal epithelium models utilizing two-dimensional (2D) human intestinal epithelial cells (hIECs) and 3D human intestinal organoids (hIOs), which mimic in vivo cellular diversity and function, in order to examine the deleterious effects of PM10 in human intestine-like in vitro models. Results: Inflamed 2D hIECs and 3D hIOs exhibited pathological features, such as inflammation, decreased intestinal markers, and defective epithelial barrier function. In addition, we found that PM10 exposure induced a more severe disturbance of peptide uptake in inflamed 2D hIECs and 3D hIOs than in control cells. This was due to the fact that it interferes with calcium signaling, protein digestion, and absorption pathways. The findings demonstrate that PM10-induced epithelial alterations contribute to the exacerbation of inflammatory disorders caused by the intestine. Conclusions: According to our findings, 2D hIEC and 3D hIO models could be powerful in vitro platforms for the evaluation of the causal relationship between PM exposure and abnormal human intestinal functions.
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Células Epiteliales , Intestinos , Humanos , Organoides , Señalización del Calcio , Inflamación , Material Particulado/efectos adversosRESUMEN
Patient-derived human induced pluripotent stem cells (iPSCs) provide a potentially useful resource for studying disease pathology and therapeutics. In this study, we generated the breast cancer patient-derived KRIBBi009-A-iPSC line from normal fibroblasts using the Sendai virus, which expressed pluripotent markers and exhibited differentiation capacity across 3 germ layers through in vitro differentiation and in vivo teratoma assay. A normal karyotype and the absence of cross-contamination of the cell lines were confirmed. Consequently, the developed iPSC line has been confirmed to be suitable for use in various studies.
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Neoplasias de la Mama , Células Madre Pluripotentes Inducidas , Humanos , Femenino , Células Madre Pluripotentes Inducidas/metabolismo , Neoplasias de la Mama/genética , Diferenciación Celular , Línea Celular , Fibroblastos/metabolismoRESUMEN
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.
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Microbioma Gastrointestinal , Células Madre Pluripotentes , Animales , Citocinas/metabolismo , Femenino , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Humanos , Mucosa Intestinal/metabolismo , Ratones , Organoides , EmbarazoRESUMEN
Many of early findings regarding intestinal stem cells (ISCs) and their niche in the human intestine have relied on colorectal cancer cell lines and labor-intensive and time-consuming mouse models. However, these models cannot accurately recapitulate the physiologically relevant aspects of human ISCs. In this study, we demonstrate a reliable and robust culture method for 3D expanding intestinal spheroids (InSexp) mainly comprising ISCs and progenitors, which can be derived from 3D human intestinal organoids (HIOs). We did functional chararcterization of InSexp derived from 3D HIOs, differentiated from human pluripotent stem cells, and optimization culture methods. Our results indicate that InSexp can be rapidly expanded and easily passaged, and show enhanced growth rates via WNT pathway activation. InSexp are capable of exponential cell expansion and cryopreservation. Furthermore, in vitro-matured HIO-derived InSexp proliferate faster than immature HIO-derived InSexp with preservation of the parental HIO characteristics. These findings may facilitate the development of scalable culture systems for the long-term maintenance of human ISCs and provide an alternative platform for studying ISC biology.
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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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Human pluripotent stem cell (hPSC)-derived organoids and cells have similar characteristics to human organs and tissues. Thus, in vitro human organoids and cells serve as a superior alternative to conventional cell lines and animal models in drug development and regenerative medicine. For a simple and reproducible analysis of the quality of organoids and cells to compensate for the shortcomings of existing experimental validation studies, a quantitative evaluation method should be developed. Here, using the GTEx database, we construct a quantitative calculation system to assess similarity to the human organs. To evaluate our system, we generate hPSC-derived organoids and cells, and detected organ similarity. To facilitate the access of our system by researchers, we develop a web-based user interface presenting similarity to the appropriate organs as percentages. Thus, this program could provide valuable information for the generation of high-quality organoids and cells and a strategy to guide proper lineage-oriented differentiation.
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Algoritmos , Diferenciación Celular/genética , Especificidad de Órganos/genética , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo , Transcriptoma/genética , Técnicas de Cultivo de Célula/métodos , Línea Celular , Perfilación de la Expresión Génica/métodos , Humanos , Organoides/citología , Células Madre Pluripotentes/citología , RNA-Seq/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
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Citocromo P-450 CYP3A , Mucosa Intestinal , Células CACO-2 , Citocromo P-450 CYP3A/metabolismo , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Organoides/metabolismoRESUMEN
Lactobacilli, which are probiotic commensal bacteria that mainly reside in the human small intestine, have attracted attention for their ability to exert health-promoting effects and beneficially modulate host immunity. However, host epithelial-commensal bacterial interactions are still largely unexplored because of limited access to human small intestinal tissues. Recently, we described an in vitro maturation technique for generating adult-like, mature human intestinal organoids (hIOs) from human pluripotent stem cells (hPSCs) that closely resemble the in vivo tissue structure and cellular diversity. Here, we established an in vitro human model to study the response to colonization by commensal bacteria using luminal microinjection into mature hIOs, allowing for the direct examination of epithelial-bacterial interactions. Lactobacillus reuteri and Lactobacillus plantarum were more likely to survive and colonize when microinjected into the lumen of mature hIOs than when injected into immature hIOs, as determined by scanning electron microscopy, colony formation assay, immunofluorescence, and real-time imaging with L plantarum expressing red fluorescent protein. The improved mature hIO-based host epithelium system resulted from enhanced intestinal epithelial integrity via upregulation of mucus secretion and tight junction proteins. Our study indicates that mature hIOs are a physiologically relevant in vitro model system for studying commensal microorganisms.
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Diferenciación Celular , Mucosa Intestinal/citología , Intestinos/citología , Lactobacillus/crecimiento & desarrollo , Organoides/citología , Células Madre Pluripotentes/citología , Células Cultivadas , Humanos , Técnicas In Vitro , Mucosa Intestinal/microbiología , Intestinos/microbiología , Organoides/microbiología , Células Madre Pluripotentes/microbiologíaRESUMEN
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. Methods: 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. Results: 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. Conclusions: 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.
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Colitis/tratamiento farmacológico , Sulfato de Dextran/toxicidad , Inflamación/prevención & control , Interleucina-2/farmacología , Mucosa Intestinal/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/química , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/patología , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Human induced pluripotent stem cells (hiPSCs) are reprogrammed from somatic cells and are regarded as promising sources for regenerative medicine and disease research. Recently, techniques for analyses of individual cells, such as single-cell RNA-Seq and mass cytometry, have been used to understand the stem cell reprogramming process in the mouse. However, the reprogramming process in hiPSCs remains poorly understood. Here we used mass cytometry to analyze the expression of pluripotency and cell cycle markers in the reprogramming of human stem cells. We confirmed that, during reprogramming, the main cell population was shifted to an intermediate population consisting of neither fibroblasts nor hiPSCs. Detailed population analyses using computational approaches, including dimensional reduction by spanning-tree progression analysis of density-normalized events, PhenoGraph, and diffusion mapping, revealed several distinct cell clusters representing the cells along the reprogramming route. Interestingly, correlation analysis of various markers in hiPSCs revealed that the pluripotency marker TRA-1-60 behaves in a pattern that is different from other pluripotency markers. Furthermore, we found that the expression pattern of another pluripotency marker, octamer-binding protein 4 (OCT4), was distinctive in the pHistone-H3high population (M phase) of the cell cycle. To the best of our knowledge, this is the first mass cytometry-based investigation of human reprogramming and pluripotency. Our analysis elucidates several aspects of hiPSC reprogramming, including several intermediate cell clusters active during the process of reprogramming and distinctive marker expression patterns in hiPSCs.
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Biomarcadores , Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Fosfatasa Alcalina/metabolismo , Antígenos de Superficie/metabolismo , Biomarcadores/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Biología Computacional , Técnica del Anticuerpo Fluorescente , Humanos , Citometría de Imagen , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Proteoglicanos/metabolismo , Factores de Transcripción SOXB1/metabolismo , Análisis de la Célula IndividualRESUMEN
Reprogramming with episomal vectors is an easy, safe, and cost-effective method to generate exogenous DNA-free (exogene-free) induced pluripotent stem cells (iPSCs). However, the genomic integration of exogenes is observed occasionally. Additionally, the removal of episomal DNA takes more than 70 days in established iPSCs. Here, we inserted the cytosine deaminase (CD) gene from yeast into episomal vectors and used them to reprogram human fibroblasts into iPSCs. These new episomal vectors (CD episomal vectors) were eliminated from the generated iPSCs as early as seven days after 5-fluorocytosine (5-FC) treatment. We also found that cells with the integration of the CD gene perished within two days of 5-FC treatment. In addition, we generated exogene-free induced neural stem cells after one passage by direct reprogramming with CD episomal vectors combined with 5-FC treatment. Conclusively, our novel method allows the rapid and easy isolation of exogene-free reprogrammed cells and can be applied to disease modeling and clinical applications.
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Calcio/metabolismo , Transdiferenciación Celular/genética , Reprogramación Celular , Células Madre Pluripotentes Inducidas/fisiología , Línea Celular , Citosina Desaminasa/genética , Fibroblastos , Flucitosina , Vectores Genéticos/genética , Humanos , Células-Madre Neurales/fisiología , Plásmidos/genéticaRESUMEN
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.
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BACKGROUND & AIMS: The development of hepatic models capable of long-term expansion with competent liver functionality is technically challenging in a personalized setting. Stem cell-based organoid technologies can provide an alternative source of patient-derived primary hepatocytes. However, self-renewing and functionally competent human pluripotent stem cell (PSC)-derived hepatic organoids have not been developed. METHODS: We developed a novel method to efficiently and reproducibly generate functionally mature human hepatic organoids derived from PSCs, including human embryonic stem cells and induced PSCs. The maturity of the organoids was validated by a detailed transcriptome analysis and functional performance assays. The organoids were applied to screening platforms for the prediction of toxicity and the evaluation of drugs that target hepatic steatosis through real-time monitoring of cellular bioenergetics and high-content analyses. RESULTS: Our organoids were morphologically indistinguishable from adult liver tissue-derived epithelial organoids and exhibited self-renewal. With further maturation, their molecular features approximated those of liver tissue, although these features were lacking in 2D differentiated hepatocytes. Our organoids preserved mature liver properties, including serum protein production, drug metabolism and detoxifying functions, active mitochondrial bioenergetics, and regenerative and inflammatory responses. The organoids exhibited significant toxic responses to clinically relevant concentrations of drugs that had been withdrawn from the market due to hepatotoxicity and recapitulated human disease phenotypes such as hepatic steatosis. CONCLUSIONS: Our organoids exhibit self-renewal (expandable and further able to differentiate) while maintaining their mature hepatic characteristics over long-term culture. These organoids may provide a versatile and valuable platform for physiologically and pathologically relevant hepatic models in the context of personalized medicine. LAY SUMMARY: A functionally mature, human cell-based liver model exhibiting human responses in toxicity prediction and drug evaluation is urgently needed for pre-clinical drug development. Here, we develop a novel human pluripotent stem cell-derived hepatocyte-like liver organoid that is critically advanced in terms of its generation method, functional performance, and application technologies. Our organoids can contribute to the better understanding of liver development and regeneration, and provide insights for metabolic studies and disease modeling, as well as toxicity assessments and drug screening for personalized medicine.
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Técnicas de Cultivo de Célula/métodos , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/citología , Hígado/citología , Organoides/citología , Acetaminofén/farmacología , Diferenciación Celular , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/inducido químicamente , Hígado/metabolismo , Organoides/efectos de los fármacos , Organoides/metabolismo , Regeneración/efectos de los fármacos , TranscriptomaRESUMEN
Spinal muscular atrophy (SMA) is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. Only â¼10% of the products of SMN2, a paralogue of SMN1, are functional full-length SMN (SMN-FL) proteins, whereas SMN2 primarily produces alternatively spliced transcripts lacking exon 7. Reduced SMN protein levels in SMA patients lead to progressive degeneration of spinal motor neurons (MNs). In this study, we report an advanced platform based on an SMN2 splicing-targeting approach for SMA drug screening and validation using an SMN2 splicing reporter cell line and an in vitro human SMA model through induced pluripotent stem cell (iPSC) technology. Through drug screening using a robust cell-based luciferase assay to quantitatively measure SMN2 splicing, the small-molecule candidate compound rigosertib was identified as an SMN2 splicing modulator that led to enhanced SMN protein expression. The therapeutic potential of the candidate compound was validated in MN progenitors differentiated from SMA patient-derived iPSCs (SMA iPSC-pMNs) as an in vitro human SMA model, which recapitulated the biochemical and molecular phenotypes of SMA, including lower levels of SMN-FL transcripts and protein, enhanced cell death, and reduced neurite length. The candidate compound exerted strong splicing correction activity for SMN2 and potently alleviated the disease-related phenotypes of SMA iPSC-pMNs by modulating various cellular and molecular abnormalities. Our combined screening platform representing a pMN model of human SMA provides an efficient and reliable drug screening system and is a promising resource for drug evaluation and the exploration of drug modes of action.
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Empalme Alternativo/efectos de los fármacos , Glicina/análogos & derivados , Modelos Neurológicos , Atrofia Muscular Espinal , Sulfonas/farmacología , Animales , Línea Celular , Glicina/farmacología , Humanos , Ratones , Ratones Transgénicos , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patología , Proteína 2 para la Supervivencia de la Neurona Motora/biosíntesis , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
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.
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Interleucina-2/farmacología , Intestinos/citología , Organoides/citología , Células Madre Pluripotentes/citología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Técnicas de Cocultivo , Humanos , Organoides/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/citologíaRESUMEN
Human intestinal organoids (hIOs) derived from human pluripotent stem cells (hPSCs) have immense potential as a source of intestines. Therefore, an efficient system is needed for visualizing the stage of intestinal differentiation and further identifying hIOs derived from hPSCs. Here, 2 fluorescent biosensors were developed based on human induced pluripotent stem cell (hiPSC) lines that stably expressed fluorescent reporters driven by intestine-specific gene promoters Krüppel-like factor 5 monomeric Cherry (KLF5mCherry) and intestine-specific homeobox enhanced green fluorescence protein (ISXeGFP). Then hIOs were efficiently induced from those transgenic hiPSC lines in which mCherry- or eGFP-expressing cells, which appeared during differentiation, could be identified in intact living cells in real time. Reporter gene expression had no adverse effects on differentiation into hIOs and proliferation. Using our reporter system to screen for hIO differentiation factors, we identified DMH1 as an efficient substitute for Noggin. Transplanted hIOs under the kidney capsule were tracked with fluorescence imaging (FLI) and confirmed histologically. After orthotopic transplantation, the localization of the hIOs in the small intestine could be accurately visualized using FLI. Our study establishes a selective system for monitoring the in vitro differentiation and for tracking the in vivo localization of hIOs and contributes to further improvement of cell-based therapies and preclinical screenings in the intestinal field.-Jung, K. B., Lee, H., Son, Y. S., Lee, J. H., Cho, H.-S., Lee, M.-O., Oh, J.-H., Lee, J., Kim, S., Jung, C.-R., Kim, J., Son, M.-Y. In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells.
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Células Madre Pluripotentes Inducidas/citología , Intestinos/citología , Organoides/citología , Animales , Técnicas Biosensibles , Diferenciación Celular/genética , Línea Celular , Sistemas de Computación , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Xenoinjertos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Mucosa Intestinal/metabolismo , Intestino Delgado/citología , Intestino Delgado/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Organoides/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Fluorescente RojaRESUMEN
Three fluorescent nucleosides-UFL, UAF, and UDAF, containing fluorene, 2-aminofluorene, and 2-dimethylaminofluorene units, respectively, covalently attached to 2'-deoxyuridine-have been incorporated into the central positions of oligodeoxynucleotides (ODNs) to examine the effects of their flanking bases (FBs) and pH on the emission properties upon hybridization with fully matched and single-base-mismatched targets. The ODN containing UFL and cytosine-FBs in the pH range from 5.5 to 8.0 and the ODN containing UDAF and cytosine-FBs under slightly acidic conditions (pH 6.0-6.5) exhibited dramatic increases in fluorescence only upon duplex formation with their fully matched target DNAs.