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High blood pressure is a major risk factor for cardiovascular disease. Our previous study confirmed that daily intake of casein hydrolysate that contained Met-Lys-Pro (MKP) can safely lower mildly elevated blood pressure. The present study aimed to evaluate the intestinal absorption differences between peptide MKP as a casein hydrolysate and synthetic MKP alone using Caco-2 cells and human iPS cell-derived small intestinal epithelial cells (hiSIECs). MKP was transported intact through Caco-2 cells and hiSIECs with permeability coefficient (Papp) values of 0.57 ± 0.14 × 10-7 and 1.03 ± 0.44 × 10-7 cm/s, respectively. This difference in Papp suggests differences in the tight junction strength and peptidase activity of each cell. Moreover, the transepithelial transport and residual ratio of intact MKP after adding casein hydrolysate containing MKP was significantly higher than that after adding synthetic MKP alone, suggesting that other peptides in casein hydrolysate suppressed MKP degradation and increased itsãtransport. These findings suggest that hiSIECs could be useful for predicting the human intestinal absorption of bioactive peptides; ingesting MKP as a casein hydrolysate may also improve MKP bioavailability.
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Caseínas , Células Epiteliales , Absorción Intestinal , Intestino Delgado , Humanos , Caseínas/metabolismo , Células CACO-2 , Absorción Intestinal/efectos de los fármacos , Intestino Delgado/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Disponibilidad Biológica , PermeabilidadRESUMEN
Background and Objectives: Despite the fact that biologic drugs have transformed inflammatory bowel disease (IBD) treatment, addressing fibrosis-related strictures remains a research gap. This study explored the roles of cytokines, macrophages, and Krüppel-like factors (KLFs), specifically KLF4, in intestinal fibrosis, as well as the interplay of KLF4 with various gut components. Materials and Methods: This study examined macrophage subtypes, their KLF4 expression, and the effects of KLF4 knockdown on macrophage polarization and cytokine expression using THP-1 monocyte models. Co-culture experiments with stromal myofibroblasts and a conditioned medium from macrophage subtype cultures were conducted to study the role of these cells in intestinal fibrosis. Human-induced pluripotent stem cell-derived small intestinal organoids were used to confirm inflammatory and fibrotic responses in the human small intestinal epithelium. Results: Each macrophage subtype exhibited distinct phenotypes and KLF4 expression. Knockdown of KLF4 induced inflammatory cytokine expression in M0, M2a, and M2c cells. M2b exerted anti-fibrotic effects via interleukin (IL)-10. M0 and M2b cells showed a high migratory capacity toward activated stromal myofibroblasts. M0 cells interacting with activated stromal myofibroblasts transformed into inflammatory macrophages, thereby increasing pro-inflammatory cytokine expression. The expression of IL-36α, linked to fibrosis, was upregulated. Conclusions: This study elucidated the role of KLF4 in macrophage polarization and the intricate interactions between macrophages, stromal myofibroblasts, and cytokines in experimental in vitro models of intestinal fibrosis. The obtained results may suggest the mechanism of fibrosis formation in clinical IBD.
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Fibrosis , Factor 4 Similar a Kruppel , Macrófagos , Humanos , Citocinas/metabolismo , Enfermedades Inflamatorias del Intestino , Macrófagos/metabolismo , Monocitos/metabolismo , Fenotipo , Células THP-1RESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is associated with various neurological symptoms, including nausea, dizziness, headache, encephalitis, and epileptic seizures. SARS-CoV-2 is considered to affect the central nervous system (CNS) by interacting with the blood-brain barrier (BBB), which is defined by tight junctions that seal paracellular gaps between brain microvascular endothelial cells (BMECs). Although SARS-CoV-2 infection of BMECs has been reported, the detailed mechanism has not been fully elucidated. METHODS: Using the original strain of SARS-CoV-2, the infection in BMECs was confirmed by a detection of intracellular RNA copy number and localization of viral particles. BMEC functions were evaluated by measuring transendothelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics, and expression levels of proinflammatory genes. BMEC signaling pathway was examined by comprehensive RNA-seq analysis. RESULTS: We observed that iPSC derived brain microvascular endothelial like cells (iPSC-BMELCs) were infected with SARS-CoV-2. SARS-CoV-2 infection resulted in decreased TEER. In addition, SARS-CoV-2 infection decreased expression levels of tight junction markers CLDN3 and CLDN11. SARS-CoV-2 infection also increased expression levels of proinflammatory genes, which are known to be elevated in patients with COVID-19. Furthermore, RNA-seq analysis revealed that SARS-CoV-2 dysregulated the canonical Wnt signaling pathway in iPSC-BMELCs. Modulation of the Wnt signaling by CHIR99021 partially inhibited the infection and the subsequent inflammatory responses. CONCLUSION: These findings suggest that SARS-CoV-2 infection causes BBB dysfunction via Wnt signaling. Thus, iPSC-BMELCs are a useful in vitro model for elucidating COVID-19 neuropathology and drug development.
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COVID-19 , Células Madre Pluripotentes Inducidas , Humanos , SARS-CoV-2 , Vía de Señalización Wnt , Células Endoteliales/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Encéfalo/irrigación sanguínea , Barrera Hematoencefálica/metabolismoRESUMEN
Macrophages play a role in nephrolithiasis, offering the possibility of developing macrophage-mediated preventive therapies. To establish a system for screening drugs that could prevent the formation of kidney stones, we aimed to develop a model using human induced pluripotent stem cell (iPSC)-derived macrophages to study phagocytosis of calcium oxalate monohydrate (COM) crystals. Human iPSCs (201B7) were cultured. CD14+ monocytes were recovered using a stepwise process that involved the use of growth factors and cytokines. These cells were then allowed to differentiate into M1 and M2 macrophages. The macrophages were co-cultured with COM crystals and used in the phagocytosis experiments. Live cell imaging and polarized light observation via super-resolution microscopy were used to visualize phagocytosis. Localization of phagocytosed COM crystals was observed using transmission electron microscopy. Intracellular fluorescence intensity was measured using imaging cytometry to quantify phagocytosis. Human iPSCs successfully differentiated into M1 and M2 macrophages. M1 macrophages adhered to the culture plate and moved COM crystals from the periphery to cell center over time, whereas M2 macrophages did not adhere to the culture plate and actively phagocytosed the surrounding COM crystals. Fluorescence assessment over a 24-h period showed that M2 macrophages exhibited higher intracellular fluorescence intensity (5.65-times higher than that of M1 macrophages at 4.5 h) and maintained this advantage for 18 h. This study revealed that human iPSC-derived macrophages have the ability to phagocytose COM crystals, presenting a new approach for studying urinary stone formation and highlighting the potential of iPSC-derived macrophages as a tool to screen nephrolithiasis-related drugs.
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Células Madre Pluripotentes Inducidas , Cálculos Renales , Humanos , Oxalato de Calcio/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos/metabolismo , Fagocitosis , Cálculos Renales/metabolismoRESUMEN
An in vitro system that evaluates pharmacokinetics in the small intestine is crucial for the development of oral drugs. We produced human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) with high drug metabolizing enzyme and drug transporter activities. However, the gene expression of our hiSIECs partially differed from that of the human small intestine, with low drug metabolizing enzyme activities. Therefore, we used air-liquid interface (ALI) culture and 5-aza-2'-deoxycytidine (5AZA)-free medium to generate hiSIECs (novel hiSIECs). Novel hiSIECs showed enhanced gene expression of drug metabolizing enzymes, such as cytochrome P450 (CYP)3A4, CYP2C9, CYP2C19, and carboxylesterase 2 that are highly expressed in the small intestine. In addition, the expression of genes involved in nutrient absorption-one of the major functions of the small intestine-also increased. The novel hiSIECs expressed ZO-1 and E-cadherin. Moreover, the novel hiSIECs exhibited a barrier function that allowed low lucifer yellow permeation. The novel hiSIECs showed high activities of CYP3A4, CYP2C9, and CYP2C19, which are abundantly expressed in the small intestine. In conclusion, the novel hiSIECs have great potential as an in vitro system to evaluate pharmacokinetics in the small intestine.
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Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Intestinos , Células Epiteliales/metabolismoRESUMEN
Most orally administered drugs exert their effects after being absorbed in the small intestine. Therefore, new drugs must undergo nonclinical pharmacokinetic evaluations in the small intestine. Enterocytes derived from human induced pluripotent stem cells (hiPSCs) are expected to be used in the evaluation system, as they reflect human intestinal characteristics more accurately; moreover, several differentiation protocols are available for these cells. However, enterocytes derived from hiPSCs have drawbacks such as time, cost, and lot-to-lot differences. Hence, to address these issues, we attempted to maintain hiPSC-derived intestinal stem cells (ISCs) that can differentiate into various intestinal cells by regulating various pathways. Although our previous attempt was partly successful, the drawbacks of elevated cost and complicated handling remained, because more than 10 factors (A 83-01, CHIR99021, epidermal growth factor, basic fibroblast growth factor, SB202190, nicotinamide, N-acetylcysteine, valproic acid, Wnt3a, R-spondin 1, and noggin) are needed to maintain ISCs. Therefore, in this study, we successfully maintained ISCs using only five factors, including growth factors. Moreover, we generated not only enterocytes but also intestinal organoids from the maintained ISCs. Thus, our novel findings provided a time-saving and cost-effective culture method for enterocytes derived from hiPSCs.
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Células Madre Pluripotentes Inducidas , Humanos , Intestinos , Intestino Delgado/metabolismo , Enterocitos/metabolismo , Diferenciación CelularRESUMEN
Patients with diabetes mellitus (DM) often experience complications such as peripheral arterial disease (PAD), which is thought to be caused by vascular damage resulting from increased oxidative stress. Dipeptidyl peptidase-4 inhibitors have been reported to reduce oxidative stress, although the exact mechanism remains unclear. This study aimed to investigate the impact of long-term (6 weeks) anagliptin treatment at a dose of 200 mg/kg/d against oxidative stress in the femoral artery of Otsuka Long-Evans Tokushima Fatty (OLETF) rats using a well-established animal model for type 2 DM. Serum toxic advanced glycation end-products concentrations and blood glucose levels after glucose loading were significantly elevated in OLETF rats compared to Long-Evans Tokushima Otsuka (LETO) rats but were significantly suppressed by anagliptin administration. Plasma glucagon-like peptide-1 concentrations after glucose loading were significantly increased in anagliptin-treated rats. Superoxide production and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in femoral arteries were significantly increased in OLETF rats compared to LETO rats but were significantly decreased by anagliptin administration. The expressions of NADPH oxidase components (p22phox in the intima region and p22phox and gp91phox in the media region) in the femoral artery were significantly increased in OLETF rats compared to LETO rats but were significantly suppressed by anagliptin administration. Furthermore, the femoral artery showed increased wall thickness in OLETF rats compared to LETO rats, but anagliptin administration reduced the thickening. This study suggests that long-term anagliptin administration can reduce oxidative stress in femoral arteries and improve vascular injury.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Pirimidinas , Lesiones del Sistema Vascular , Humanos , Ratas , Animales , Arteria Femoral , Lesiones del Sistema Vascular/tratamiento farmacológico , Ratas Endogámicas OLETF , Ratas Long-Evans , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , GlucosaRESUMEN
The small intestine, which plays a crucial role in the absorption and metabolism of drugs and foods, serves as a target organ for drug-induced toxicity and immune interactions with functional foods and intestinal bacteria. Current alternative models of the human small intestine, such as Caco-2 cells and experimental animals, have limitations due to variations in the expression levels of metabolic enzymes, transporters, and receptors. This study presents investigations into the utility of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) for pharmacokinetic, toxicological, and immunological studies, respectively. While hiSIECs displayed small intestinal epithelial cell characteristics and barrier function, they demonstrated pharmacokinetic properties such as cytochrome P450 3A4/5 activity equivalent to human primary enterocytes and stable P-glycoprotein activity. These cells also demonstrated potential for assessing two forms of intestinal toxicity caused by anticancer drugs and gamma-secretase inhibitors, displaying immune responses mediated by toll-like and fatty acid receptors while serving as an inflammatory gut model through the addition of tumor necrosis factor alpha and interferon gamma. Overall, hiSIECs hold promise as an in vitro model for assessing pharmacokinetics, toxicity, and effects on the intestinal immunity of pharmaceuticals, functional foods, supplements, and intestinal bacteria.
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Células Madre Pluripotentes Inducidas , Animales , Humanos , Células CACO-2 , Células Madre Pluripotentes Inducidas/metabolismo , Diferenciación Celular , Células Epiteliales/metabolismo , Intestino Delgado/metabolismo , Proteínas Portadoras/metabolismo , Mucosa Intestinal/metabolismoRESUMEN
The development of models for predicting hepatotoxicity is warranted, as the hepatotoxicity risk of 38-51% of compounds is undetectable in nonclinical studies. Cholestatic drug-induced liver injury (DILI) is a condition in which bile acids are abnormally excreted into the capillary bile canaliculi and are accumulated in hepatocytes, caused by the inhibition of bile salt export pump (BSEP), a transporter that is mainly associated with excretion of bile acids. Although laboratory animals are used as models, the use of human-derived cells is required owing to species differences. Unfortunately, primary human hepatocytes (PHHs) show rapid loss of function in culture and difficulties in forming bile canaliculi. Therefore, we aimed to develop an in vitro culture method for the efficient formation of bile canaliculi and for assessing the function of BSEP in PHHs. Here, PHHs were cultured from 1 h after thawing to day 2 with Z-VAD-FMK, a total caspase inhibitor, and RevitaCell™ supplement, an irreversible Rho-associated coiled-coil forming kinase (ROCK) inhibitor, in combination with RM-101. The PHHs formed bile canaliculi and showed BSEP function on day 6 of culture. Our findings suggest that cultured PHHs may improve the prediction accuracy of the risks of cholestatic DILI-contained toxicity on bile canaliculi.
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Efforts have been made to replace animal experiments in safety evaluations, including in vitro-based predictions of human internal exposures, such as predicting peak plasma concentration (Cmax) values for xenobiotics and comparing these values with in vitro-based toxicity endpoints. Herein, the authors predicted the Cmax values of food-related compounds in humans based on existing and novel in vitro techniques. In this study, 20 food-related compounds, which have been previously reported in human pharmacokinetic or toxicokinetic studies, were evaluated. Human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIEC) and Caco-2 cells, HepaRG cells, equilibrium dialysis of human plasma, and LLC-PK1 cell monolayer were used to assess intestinal absorption and availability, hepatic metabolism, unbound plasma fraction, and secretion and reabsorption in renal tubular cells, respectively. After conversion of these parameters into human kinetic parameters, the plasma concentration profiles of these compounds were predicted using in silico methods, and the obtained Cmax values were found to be between 0.017 and 183 times the reported Cmax values. When the in silico-predicted parameters were modified with in vitro data, the predicted Cmax values came within 0.1-10 times the reported values because the metabolic activities of hiPSC-SIECs, such as uridine 5'-diphospho-glucuronosyl transferase, are more similar to those of human primary enterocytes. Thus, combining in vitro test results with the plasma concentration simulations resulted in more accurate and transparent predictions of Cmax values of food-related compounds than those obtained using in silico-derived predictions alone. This method facilitates accurate safety evaluation without the need for animal experiments.
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Células Madre Pluripotentes Inducidas , Animales , Humanos , Simulación por Computador , Células CACO-2 , Administración Oral , Alimentos , Modelos BiológicosRESUMEN
Our previous study showed that the flotillin level is decreased in the blood of patients with Alzheimer's disease (AD) when compared to that of patients with non-AD and vascular dementia; however, the molecular mechanism remains to be determined. In this study, to elucidate whether Aß accumulation in the brain has an effect on the blood flotillin level, we used our previously established blood-brain barrier (BBB) culture model using microvascular endothelial cells obtained from human induced pluripotent stem cells (iBMECs) and astrocytes prepared from rat cortex. In this BBB model with iBMECs plated on the upper compartment (blood side) and astrocytes plated on the lower compartment (brain side), the trans-endothelial electrical resistance values are high (over 1500 Ωm2) and stable during experiments. We found that the addition of Aß42 (0.5 and 2 µM) to the brain side significantly reduced the level of flotillin secreted by iBMECs on the blood side. The level of basic fibroblast growth factor (FGF-2) in the brain side was significantly reduced by Aß42 treatment, and was accompanied by a reduction in the level of phosphorylation of the fibroblast growth factor receptor in iBMECs. The brain-side Aß42 treatment-induced reduction of flotillin secretion into the blood side was restored in a dose-dependent manner by the addition of FGF-2 into the brain side. These results indicated that Aß accumulation in the brain side reduced FGF-2 release from astrocytes, which attenuated FGF-2-mediated iBMECs signaling via the FGF-2 receptor, and thereby reduced flotillin secretion from iBMECs on the blood side. Our findings revealed a novel signaling pathway crossing the BBB from the brain side to the blood side, which is different from the classical intramural periarterial drainage or lymphatic-system-to-blood pathway.
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Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Humanos , Animales , Ratas , Barrera Hematoencefálica/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Células Endoteliales/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Encéfalo/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismoRESUMEN
Xanthine oxidoreductase exists both intracellularly and extracellularly and induces vascular injury by producing reactive oxygen species (ROS). Here, we investigated the effects and mechanism of action of topiroxostat, a xanthine oxidase inhibitor, on ROS using an animal model of type 1 diabetes with persistent hyperglycemia. Six-week-old male Sprague-Dawley rats were administered 50 mg/kg streptozotocin to induce diabetes; at 8 weeks of age, animals were administered topiroxostat (0.3, 1, or 3 mg/kg) for 2 weeks through mixed feeding after which the aorta was sampled. The production of superoxide, a type of ROS, was measured by chemiluminescence and dihydroethidium staining. Cytotoxicity was evaluated by nitrotyrosine staining. Topiroxostat at 3 mg/kg significantly decreased blood urea nitrogen, e-selectin, urinary malondialdehyde, and the urinary albumin/creatinine ratio compared with the streptozotocin group. Superoxide production by xanthine oxidase anchored to the cell membrane was significantly decreased by topiroxostat at both 1 mg/kg and 3 mg/kg compared with the streptozotocin group. Dihydroethidium staining revealed no significant effect of topiroxostat administration on superoxide production. The fluorescence intensity of nitrotyrosine staining was significantly suppressed by 3 mg/kg topiroxostat. Topiroxostat was found to inhibit the production of ROS in the thoracic aorta and suppress vascular endothelial damage. The antioxidant effect of topiroxostat appears to be exerted via the inhibition of anchored xanthine oxidase.
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Diabetes Mellitus Experimental , Xantina Oxidasa , Ratas , Masculino , Animales , Estreptozocina , Especies Reactivas de Oxígeno , Diabetes Mellitus Experimental/tratamiento farmacológico , Superóxidos , Ratas Sprague-Dawley , Estrés Oxidativo , AortaRESUMEN
Brain microvascular endothelial cells (BMECs) constitute the blood-brain barrier (BBB), which prevents the transfer of substances into the brain. Recently, in vitro BBB models using human-induced pluripotent stem (iPS) cell-derived brain microvascular endothelial-like cells (iBMELCs) have been created. However, it is suggested that iBMELCs differentiated by the existing methods are different from the BMECs that occur in vivo. This study aimed to establish iBMELCs generated via human iPS cell-derived endothelial progenitor cells (iEPCs) (E-iBMELCs). Expanded and cryopreserved iEPCs were thawed and differentiated into mature endothelial cells under various conditions. Intercellular barriers were significantly enhanced in E-iBMELCs using a B-27 supplement, transforming growth factor-ß receptor inhibitor, and laminin 511 fragment. Expression of the endothelial cell markers was higher in the E-iBMELCs generated in this study compared with conventional methods. In addition, E-iBMELCs expressed P-glycoprotein. E-iBMELCs developed in this study will significantly contribute to drug discovery for neurodegenerative diseases and might elucidate the pathogenesis of neurodegenerative diseases associated with BBB disruption.
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Hepatitis B virus (HBV) specifically infects human hepatocytes and increases the risks of cirrhosis and liver cancer. Currently, nucleic acid analogs are the main therapeutics for chronic hepatitis caused by HBV infection. Although nucleic acid analogs can eliminate HBV DNA by inhibiting HBV reverse transcriptase, they cannot lead to negative conversion of covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg). In this study, we revealed that the antifilarial drug ivermectin suppresses HBV production by a different mechanism from the nucleic acid analog entecavir or Na+ taurocholate co-transporting polypeptide-mediated entry inhibitor cyclosporin A. Ivermectin reduced the levels of several HBV markers, including HBsAg, in HBV-infected human hepatocellular carcinoma cells (HepG2-hNTCP-C4 cells) and humanized mouse hepatocytes (PXB hepatocytes). In addition, ivermectin significantly decreased the expression of HBV core protein and the nuclear transporter karyopherin α2 (KPNA2) in the nuclei of HepG2-hNTCP-C4 cells. Furthermore, depletion of KPNA1-6 suppressed the production of cccDNA. These results suggest that KPNA1-6 is involved in the nuclear import of HBV and that ivermectin suppresses the nuclear import of HBV by inhibiting KPNA2. This study demonstrates the potential of ivermectin as a novel treatment for hepatitis B.
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Hepatitis B Crónica , Hepatitis B , Ratones , Animales , Humanos , Virus de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Ivermectina/farmacología , ADN Circular/metabolismo , ADN Viral/metabolismo , Replicación Viral/genética , alfa Carioferinas/metabolismoRESUMEN
Introduction: Ulcerative colitis (UC) is an inflammatory bowel disease characterized by repeated remissions and relapses. Immunosuppressive drugs have facilitated the induction and maintenance of remission in many patients with UC. However, immunosuppressive drugs cannot directly repair impaired intestinal mucosa and are insufficient for preventing relapse. Therefore, new treatment approaches to repair the damaged epithelium in UC have been attempted through the transplantation of intestinal organoids, which can be differentiated into mucosa by embedding in Matrigel, generated from patient-derived intestinal stem cells. The method, however, poses the challenge of yielding sufficient cells for UC therapy, and patient-derived cells might already have acquired pathological changes. In contrast, human induced pluripotent stem (iPS) cells generated from healthy individuals are infinitely proliferated and can be differentiated into target cells. Recently developed human iPS cell-derived intestinal organoids (HIOs) aim to generate organoids that closely resemble the adult intestine. However, no study till date has reported HIOs injected into in vivo inflammatory models, and it remains unclear whether HIOs with cells that closely resemble the adult intestine or with intestinal stem cells retain the better ability to repair tissue in colitis. Methods: We generated two types of HIOs via suspension culture with and without small-molecule compounds: HIOs that include predominantly more intestinal stem cells [HIO (A)] and those that include predominantly more intestinal epithelial and secretory cells [HIO (B)]. We examined whether the generated HIOs engrafted in vivo and compared their ability to accelerate recovery of the damaged tissue. Results: Findings showed that the HIOs expressed intestinal-specific markers such as caudal-type homeobox 2 (CDX2) and villin, and HIOs engrafted under the kidney capsules of mice. We then injected HIOs into colitis-model mice and found that the weight and clinical score of the mice injected with HIO (A) recovered earlier than that of the mice in the sham group. Further, the production of mucus and the expression of cell proliferation markers and tight junction proteins in the colon tissues of the HIO (A) group were restored to levels similar to those observed in healthy mice. However, neither HIO (A) nor HIO (B) could be engrafted into the colon. Conclusions: Effective cell therapy should directly repair tissue by engraftment at the site of injury. However, the difference in organoid property impacting the rate of tissue repair in transplantation without engraftment observed in the current study should be considered a critical consideration in the development of regenerative medicine using iPS-derived organoids.
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Currently, there are many challenges in the culture of human induced pluripotent stem (iPS) cell-derived intestinal organoids (HIOs) for use in drug discovery, disease research, and regenerative medicine. For example, the main culture method, embedding culture, makes industrial large-scale culture difficult, and Matrigel, which is used for almost all HIO cultures, is not respected for its application in regenerative medicine. To overcome these challenges, we herein propose a new culture method using low concentrations of natural polysaccharides in a suspension culture. In the present study, five natural polysaccharides free from heterologous animal-derived components were used, and HIOs were successfully cultured in suspension with FP001 and FP003, which are microbial exopolysaccharide analogs of gellan gum. The fabricated HIOs were similar to living intestinal tracts with respect to their gene expression, microstructure, and protein expression. The observed activities of the drug metabolizing enzymes and drug transporters in the generated HIOs suggested that they have pharmacokinetic functions. We believe that suspension culture of HIOs using FP001 or FP003 can be widely applied to not only drug discovery research but also disease research and regenerative medicine.
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Células Madre Pluripotentes Inducidas , Organoides , Animales , Diferenciación Celular/genética , Humanos , Mucosa Intestinal/metabolismo , Intestinos , Polisacáridos/metabolismo , Compuestos de AzufreRESUMEN
The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the liver MPS model has not been tested in the preclinical stage because of its obscure utility. It can overcome the major problem of conventional systems-rapid loss of mitochondrial activity in cultured hepatocytes due to limited oxygen supply-by supplying oxygen to cultured hepatocytes using a perfusion device. In this study, we developed a new perfusion culture system that can detect mitochondrial toxicity. Primary mouse hepatocytes were cultured under perfusion condition for 48 hr. The hepatocytes showed increased oxygen consumption and reduced lactate release. These results indicated that the ATP-production pathway was switched from glycolysis to mitochondrial oxidative phosphorylation in the perfusion culture system. Furthermore, ATP levels were considerably reduced in the perfusion culture system after exposure to phenformin, a mitochondrial complex I inhibitor. To summarize, the perfusion culture system could improve the mitochondrial activity in primary mouse hepatocytes, and thus, has potential implications in the detection of mitochondrial toxicity.
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Hepatocitos , Fosforilación Oxidativa , Animales , Células Cultivadas , Glucólisis , Hígado/metabolismo , Ratones , Consumo de Oxígeno , PerfusiónRESUMEN
Background and Objectives: Acetylsalicylic acid (ASA) is widely used for preventing cerebrovascular and cardiovascular diseases. Gastrointestinal (GI) tract injury is one of the major complications of aspirin use, potentially leading to severe GI bleeding. However, no drugs for preventing aspirin-induced small intestinal injury have been developed. The aim of this study was to establish a human experimental model for investigating aspirin-induced small intestinal mucosal injury. In addition, we evaluated the protective effect of Irsogladine against aspirin-induced small intestinal mucosal injury using human induced pluripotent stem cell-derived 2D monolayer crypt-villus structural small intestine (2D-hiPSC-SI). Materials and Methods: Human iPS cell-derived intestinal organoids were seeded and cultured in Air-liquid interface. The permeability of 2D-hiPSC-SI was evaluated using Lucifer yellow. Changes in structure and mucosal permeability of 2D-hiPSC-SI after addition of aspirin were confirmed over time, and changes in intestinal epithelium-related markers were evaluated by real-time qPCR and Immunofluorescence staining. The effect of Irsogladine on prevention of aspirin mucosal injury was examined by adding Irsogladine to the culture medium. Results: Cultured 2D-hiPSC-SI showed multi-lineage differentiation into small intestinal epithelium comprised of absorptive cells, goblet cells, enteroendocrine cells, and Paneth cells, which express CD10, MUC2, chromogranin A, and lysozyme, respectively. RNA in situ hybridization revealed intestinal stem cells that express Lgr5. ASA administration induced an increase in the mucosal permeability of 2D-hiPSC-SI. ASA-injured 2D-hiPSC-SI showed decreased mRNA expression of multi-lineage small intestinal cell markers as well as intestinal stem cell marker Lgr5. Administration of Irsogladine on the basal side of the 2D-hiPSC-SI resulted in significant increases in Mki67 and Muc2 mRNA expression by 2D-hiPSCs at 48 h compared with the control group. Administration of 400 µg/mL Irsogladine to the ASA-induced small intestinal injury model resulting in significantly decreased mucosal permeability of 2D-hiPSC-SI. In immunofluorescence staining, Irsogladine significantly increased the fluorescence intensity of MUC2 under normal conditions and administration of 400 µg/mL ASA. Conclusions: we established a novel ASA-induced small intestinal injury model using human iPSC-derived small intestine. Irsogladine maintains mucosal permeability and goblet cell differentiation against ASA-induced small intestinal injury.
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Aspirina , Células Madre Pluripotentes Inducidas , Humanos , Aspirina/efectos adversos , Intestino Delgado/metabolismo , ARN Mensajero/metabolismoRESUMEN
Pharmacokinetic prediction after oral ingestion is important for quantitative risk assessment of food-derived compounds. To evaluate the utility of human intestinal absorption prediction, we compared the membrane permeability and metabolic activities of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIECs) with Caco-2 cells or human primary enterocytes (hPECs). We found that membrane permeability in hiPSC-SIECs had better predictivity than that in Caco-2 cells against 21 drugs with known human intestinal availability (r = 0.830 and 0.401, respectively). Membrane permeability in hiPSC-SIECs was only 0.019-0.25-fold as compared with that in Caco-2 cells for 7 in 15 food-derived compounds, primarily those that were reported to undergo glucuronidation metabolism. The metabolic rates of the glucuronide conjugate were similar or higher in hiPSC-SIECs as compared with hPECs but lower in Caco-2 cells. Expression levels of UDP-glucuronosyltransferase (UGT) isoform mRNA in hiPSC-SIECs were similar or higher as compared with hPECs. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism. SIGNIFICANCE STATEMENT: Gastrointestinal absorption is an important step for predicting the internal exposure of food-derived compounds. This research revealed that human induced pluripotent stem cell-derived small intestinal cells (hiPSC-SIECs) had better predictivity of intestinal availability than Caco-2 cells; furthermore, the metabolic rates of UDP-glucuronosyltransferase (UGT) substrates of hiPSC-SIECs were closer to those of human primary enterocytes than those of Caco-2 cells. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism.
Asunto(s)
Permeabilidad de la Membrana Celular , Células Epiteliales/metabolismo , Glucuronosiltransferasa/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Intestino Delgado/metabolismo , Células CACO-2 , Eritrocitos/metabolismo , Alimentos , Glucurónidos/metabolismo , Humanos , Absorción Intestinal , Intestino Delgado/citología , Preparaciones Farmacéuticas/metabolismo , Valor Predictivo de las PruebasRESUMEN
Nonalcoholic steatohepatitis (NASH), the aggressive form of the most common chronic liver disease nonalcoholic fatty liver disease, is characterized by inflammation and damage in the liver. Although hepatocyte injury and cell death have been identified as cardinal pathological features of NASH, its pathogenesis has not yet been elucidated in detail. Immortalized cell lines and primary cultured cells have been used as in vitro models of NASH. However, these cells have several disadvantages, such as specialized characteristics by immortalization or limited growth potential. To overcome these difficulties and develop a strategy to analyze the pathology of NASH, we employed hepatocyte-like cells differentiated from human induced pluripotent stem cells (hiPSC-HLCs) as an in vitro model of NASH to clarify the intracellular effects of glyceraldehyde-derived advanced glycation end-products (AGEs), also named toxic AGEs (TAGE). The viability of hiPSC-HLCs decreased with the accumulation of TAGE in the cells, which was consistent with previous findings on human hepatocellular carcinoma cells and human primary cultured hepatocytes. In addition, the TAGE accumulation up-regulated the expression of inflammation-related genes (interleukin 6, interleukin 8, and monocyte chemoattractant protein-1) in hiPSC-HLCs. These results indicated that the accumulation of TAGE induced hiPSC-HLC cytotoxicity and inflammation, which are features of the pathology of NASH. Therefore, we suggest the use of hiPSC-HLCs as an important strategy for analyses of the pathology of NASH.