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1.
Nat Commun ; 15(1): 2983, 2024 Apr 06.
Article in English | MEDLINE | ID: mdl-38582860

ABSTRACT

Akkermansia muciniphila has received great attention because of its beneficial roles in gut health by regulating gut immunity, promoting intestinal epithelial development, and improving barrier integrity. However, A. muciniphila-derived functional molecules regulating gut health are not well understood. Microbiome-secreted proteins act as key arbitrators of host-microbiome crosstalk through interactions with host cells in the gut and are important for understanding host-microbiome relationships. Herein, we report the biological function of Amuc_1409, a previously uncharacterised A. muciniphila-secreted protein. Amuc_1409 increased intestinal stem cell (ISC) proliferation and regeneration in ex vivo intestinal organoids and in vivo models of radiation- or chemotherapeutic drug-induced intestinal injury and natural aging with male mice. Mechanistically, Amuc_1409 promoted E-cadherin/ß-catenin complex dissociation via interaction with E-cadherin, resulting in the activation of Wnt/ß-catenin signaling. Our results demonstrate that Amuc_1409 plays a crucial role in intestinal homeostasis by regulating ISC activity in an E-cadherin-dependent manner and is a promising biomolecule for improving and maintaining gut health.


Subject(s)
Verrucomicrobia , beta Catenin , Male , Mice , Animals , beta Catenin/metabolism , Verrucomicrobia/metabolism , Intestines , Cadherins/metabolism , Akkermansia
2.
Nat Commun ; 15(1): 799, 2024 Jan 27.
Article in English | MEDLINE | ID: mdl-38280855

ABSTRACT

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.


Subject(s)
Intestines , Pluripotent Stem Cells , Humans , Reproducibility of Results , Intestinal Mucosa , Organoids , Cell Differentiation
3.
Adv Healthc Mater ; 12(12): e2202371, 2023 05.
Article in English | MEDLINE | ID: mdl-36652539

ABSTRACT

Enhancing cardiomyocyte (CM) maturation by topographical cues is a critical issue in cardiac tissue engineering. Thus far, single-scale topographies with a broad range of feature shapes and dimensions have been utilized including grooves, pillars, and fibers. This study reports for the first time a hierarchical structure composed of nano-pillars (nPs) on micro-wrinkles (µWs) for effective maturation of CMs. Through capillary force lithography followed by a wrinkling process, vast size ranges of topographies are fabricated, and the responses of CMs are systematically investigated. Maturation of CMs on the hierarchical structures is highly enhanced compared to a single-scale topography: cardiac differentiation of H9C2s (rat cardiomyocytes) on the hierarchical topography is ≈ 2.8 and ≈ 1.9 times higher than those consisting of single-scale µWs and nPs. Both nPs and µWs have important roles in cardiac maturation, and the aspect ratio (height/diameter) of the nPs and the wavelength of the µWs are important in CM maturation. This enhancement is caused by strong focal adhesion and nucleus mediated mechanotransduction of CMs from the confinement effects of the different wavelengths of µWs and the cellular membrane protrusion on the nPs. This study demonstrates how a large family of hierarchical structures is used for cardiac maturation.


Subject(s)
Mechanotransduction, Cellular , Myocytes, Cardiac , Rats , Animals , Tissue Engineering/methods , Cell Differentiation
4.
Gut Microbes ; 14(1): 2121580, 2022.
Article in English | MEDLINE | ID: mdl-36130031

ABSTRACT

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.


Subject(s)
Gastrointestinal Microbiome , Pluripotent Stem Cells , Animals , Cytokines/metabolism , Female , Glutamic Acid/metabolism , Glutamic Acid/pharmacology , Humans , Intestinal Mucosa/metabolism , Mice , Organoids , Pregnancy
5.
Int J Stem Cells ; 15(1): 104-111, 2022 Feb 28.
Article in English | MEDLINE | ID: mdl-35220296

ABSTRACT

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.

6.
ISME J ; 16(5): 1205-1221, 2022 05.
Article in English | MEDLINE | ID: mdl-34972816

ABSTRACT

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.


Subject(s)
Colorectal Neoplasms , Microbiota , Ubiquitin-Protein Ligases/metabolism , Histocompatibility Antigens/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Propionates , Up-Regulation
7.
Front Vet Sci ; 8: 587659, 2021.
Article in English | MEDLINE | ID: mdl-34604364

ABSTRACT

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.

8.
Nat Commun ; 12(1): 4492, 2021 07 23.
Article in English | MEDLINE | ID: mdl-34301945

ABSTRACT

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.


Subject(s)
Algorithms , Cell Differentiation/genetics , Organ Specificity/genetics , Organoids/metabolism , Pluripotent Stem Cells/metabolism , Transcriptome/genetics , Cell Culture Techniques/methods , Cell Line , Gene Expression Profiling/methods , Humans , Organoids/cytology , Pluripotent Stem Cells/cytology , RNA-Seq/methods , Reverse Transcriptase Polymerase Chain Reaction
9.
Anal Chem ; 2021 Jun 16.
Article in English | MEDLINE | ID: mdl-34132523

ABSTRACT

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) hold unprecedented promise for basic biology and translational applications. However, developing a quantitative method to evaluate the epithelial cell membrane integrity of HIOs as an in vitro intestinal barrier model is a major challenge because of their complex three-dimensional (3D) structure. In this study, we developed an impedance system to measure the change in electrical resistance of 3D HIOs depending on the integrity of the intestinal epithelial cell membrane, which can reflect functionality and maturity. The expression of intestinal maturation- and tight junction-related markers was significantly higher in HIOs matured in vitro by treatment with IL-2 than in control HIOs. Analysis of gap junction size indicated that mature HIOs have greater integrity, with approximately 30% more compact gaps than immature HIOs. We designed a multi-microchannel system controlled by the inhalation pressure where the HIO is loaded, which enhances the stability and sensitivity of the impedance signal. We demonstrated the applicability of the impedance system by showing the difference in resistance between control and mature HIOs, reflecting the expression of tight junction proteins and their maturation status. We also validated the impedance system by monitoring its resistance in real time during junctional damage to HIOs induced by a digestive agent. In summary, we suggest a quantitative method to directly quantify the physiological changes in complex 3D organoid structures based on impedance spectroscopy, which can be applied to noninvasively monitor live cells and therefore enable their use in subsequent experiments.

10.
Sci Adv ; 7(23)2021 06.
Article in English | MEDLINE | ID: mdl-34078609

ABSTRACT

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.


Subject(s)
Cytochrome P-450 CYP3A , Intestinal Mucosa , Caco-2 Cells , Cytochrome P-450 CYP3A/metabolism , Epithelial Cells/metabolism , Humans , Intestinal Mucosa/metabolism , Organoids/metabolism
11.
Theranostics ; 10(11): 5048-5063, 2020.
Article in English | MEDLINE | ID: mdl-32308767

ABSTRACT

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.


Subject(s)
Colitis/drug therapy , Dextran Sulfate/toxicity , Inflammation/prevention & control , Interleukin-2/pharmacology , Intestinal Mucosa/drug effects , Phosphatidylinositol 3-Kinases/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Colitis/chemically induced , Colitis/metabolism , Colitis/pathology , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Inflammation/etiology , Inflammation/metabolism , Inflammation/pathology , Intestinal Mucosa/metabolism , Male , Mice , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism
13.
J Biol Chem ; 294(49): 18547-18556, 2019 12 06.
Article in English | MEDLINE | ID: mdl-31570522

ABSTRACT

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.


Subject(s)
Biomarkers , Gene Expression Regulation , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Alkaline Phosphatase/metabolism , Antigens, Surface/metabolism , Biomarkers/metabolism , Cell Cycle/genetics , Cell Cycle/physiology , Cell Line , Cellular Reprogramming/genetics , Cellular Reprogramming/physiology , Computational Biology , Fluorescent Antibody Technique , Humans , Image Cytometry , Nanog Homeobox Protein/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Phenotype , Proteoglycans/metabolism , SOXB1 Transcription Factors/metabolism , Single-Cell Analysis
14.
Exp Mol Med ; 51(7): 1-12, 2019 07 19.
Article in English | MEDLINE | ID: mdl-31324753

ABSTRACT

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.


Subject(s)
Calcium/metabolism , Cell Transdifferentiation/genetics , Cellular Reprogramming , Induced Pluripotent Stem Cells/physiology , Cell Line , Cytosine Deaminase/genetics , Fibroblasts , Flucytosine , Genetic Vectors/genetics , Humans , Neural Stem Cells/physiology , Plasmids/genetics
15.
J Clin Med ; 8(7)2019 Jul 04.
Article in English | MEDLINE | ID: mdl-31277507

ABSTRACT

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.

16.
J Biol Eng ; 13: 15, 2019.
Article in English | MEDLINE | ID: mdl-30809271

ABSTRACT

BACKGROUND: Cardiac fibrosis is the most common pathway of many cardiac diseases. To date, there has been no suitable in vitro cardiac fibrosis model that could sufficiently mimic the complex environment of the human heart. Here, a three-dimensional (3D) cardiac sphere platform of contractile cardiac microtissue, composed of human embryonic stem cell (hESC)-derived cardiomyocytes (CMs) and mesenchymal stem cells (MSCs), is presented to better recapitulate the human heart. RESULTS: We hypothesized that MSCs would develop an in vitro fibrotic reaction in response to treatment with transforming growth factor-ß1 (TGF-ß1), a primary inducer of cardiac fibrosis. The addition of MSCs improved sarcomeric organization, electrophysiological properties, and the expression of cardiac-specific genes, suggesting their physiological relevance in the generation of human cardiac microtissue model in vitro. MSCs could also generate fibroblasts within 3D cardiac microtissues and, subsequently, these fibroblasts were transdifferentiated into myofibroblasts by the exogenous addition of TGF-ß1. Cardiac microtissues displayed fibrotic features such as the deposition of collagen, the presence of numerous apoptotic CMs and the dissolution of mitochondrial networks. Furthermore, treatment with pro-fibrotic substances demonstrated that this model could reproduce key molecular and cellular fibrotic events. CONCLUSIONS: This highlights the potential of our 3D cardiac microtissues as a valuable tool for manifesting and evaluating the pro-fibrotic effects of various agents, thereby representing an important step forward towards an in vitro system for the prediction of drug-induced cardiac fibrosis and the study of the pathological changes in human cardiac fibrosis.

17.
Stem Cells Dev ; 28(7): 438-453, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30667343

ABSTRACT

Spinal muscular atrophy (SMA) is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. Only ∼10% 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.


Subject(s)
Alternative Splicing/drug effects , Glycine/analogs & derivatives , Models, Neurological , Muscular Atrophy, Spinal , Sulfones/pharmacology , Animals , Cell Line , Glycine/pharmacology , Humans , Mice , Mice, Transgenic , Muscular Atrophy, Spinal/drug therapy , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/pathology , Survival of Motor Neuron 2 Protein/biosynthesis , Survival of Motor Neuron 2 Protein/genetics
18.
Nat Commun ; 9(1): 3039, 2018 08 02.
Article in English | MEDLINE | ID: mdl-30072687

ABSTRACT

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.


Subject(s)
Interleukin-2/pharmacology , Intestines/cytology , Organoids/cytology , Pluripotent Stem Cells/cytology , Biomarkers/metabolism , Cell Differentiation/drug effects , Coculture Techniques , Humans , Organoids/drug effects , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , T-Lymphocytes/cytology
19.
FASEB J ; 32(1): 111-122, 2018 01.
Article in English | MEDLINE | ID: mdl-28855280

ABSTRACT

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.


Subject(s)
Induced Pluripotent Stem Cells/cytology , Intestines/cytology , Organoids/cytology , Animals , Biosensing Techniques , Cell Differentiation/genetics , Cell Line , Computer Systems , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Heterografts , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Intestinal Mucosa/metabolism , Intestine, Small/cytology , Intestine, Small/metabolism , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Organoids/metabolism , Real-Time Polymerase Chain Reaction , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Red Fluorescent Protein
20.
Mol Biosyst ; 13(8): 1565-1574, 2017 Jul 25.
Article in English | MEDLINE | ID: mdl-28671202

ABSTRACT

Cardiomyocyte (CM) differentiation techniques for generating adult-like mature CMs remain imperfect, and the plausible underlying mechanisms remain unclear; however, there are a number of current protocols available. Here, to explore the mechanisms controlling cardiac differentiation, we analyzed the genome-wide transcription dynamics occurring during the differentiation of human pluripotent stem cells (hPSCs) into CMs using embryoid body (EB) formation. We optimized and updated the protocol to efficiently generate contracting CMs from hPSCs by adding fetal bovine serum (FBS) as a medium supplement, which could have a significant impact on the efficiency of cardiac differentiation. To identify genes, biological processes, and pathways involved in the cardiac differentiation of hPSCs, integrative and comparative analyses of the transcriptome profiles of differentiated CMs from hPSCs and of control CMs of the adult human heart (CM-AHH) were performed using gene ontology, functional annotation clustering, and pathway analyses. Several genes commonly regulated in the differentiated CMs and CM-AHH were enriched in pathways related to cell cycle and nucleotide metabolism. Strikingly, we found that current differentiation protocols did not promote sufficient expression of genes involved in oxidative phosphorylation to differentiate CMs from hPSCs compared to the expression levels in CM-AHH. Therefore, to obtain mature CMs similar to CM-AHH, these deficient pathways in CM differentiation, such as energy-related pathways, must be augmented prior to use for in vitro and in vivo applications. This approach opens up new avenues for facilitating the utilization of hPSC-derived CMs in biomedical research, drug evaluation, and clinical applications for patients with cardiac failure.


Subject(s)
Culture Media/pharmacology , Embryoid Bodies/metabolism , Myocytes, Cardiac/metabolism , Pluripotent Stem Cells/metabolism , Transcriptome , Animals , Cattle , Cell Cycle/drug effects , Cell Differentiation , Cell Line , Culture Media/chemistry , Embryoid Bodies/cytology , Feeder Cells/cytology , Fetus , Fibroblasts/cytology , Gene Expression Profiling , Gene Ontology , Genome, Human , Humans , Mice , Microarray Analysis , Molecular Sequence Annotation , Myocytes, Cardiac/cytology , Pluripotent Stem Cells/cytology
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