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CYP2D6 variants contain various single nucleotide polymorphisms as well as differing levels of metabolic activity. Among these, one of the less active variants CYP2D6*10 (100C > T) is the most prevalent mutation in East Asians, including Japanese. This mutation leads to an amino acid substitution from proline to serine, which reduces the stability of CYP2D6 and consequently decreases its metabolic activity. In this study, we used a genome editing technology called the Precise Integration into Target Chromosome (PITCh) system to stably express six drug-metabolizing enzymes (CYP3A4, POR, uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), CYP1A2, CYP2C19, CYP2C9, and CYP2D6*10) in HepG2 (CYP2D6*10 KI-HepG2) cells to examine the effect of CYP2D6*10 on drug metabolism prediction. The protein expression levels of CYP2D6 in CYP2D6*10 KI-HepG2 cells were reduced relative to those in the CYP3A4-POR-UGT1A1-CYP1A2-CYP2C19-CYP2C9-CYP2D6 knock-in-HepG2 (CYPs-UGT1A1 KI-HepG2) cells. Consistent with the CYP2D6 protein expression results, CYP2D6 metabolic activity in CYP2D6*10 KI-HepG2 cells was reduced relative to CYPs-UGT1A1 KI-HepG2 cells. We successfully generated CYP2D6*10 KI-HepG2 cells with highly expressed, functional CYP2D6*10, as well as CYP1A2, 2C9, 2C19 and 3A4. CYP2D6*10 KI-HepG2 cells could be an invaluable model for hepatic metabolism and hepatotoxicity studies in East Asians, including Japanese.
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Citocromo P-450 CYP2D6 , Hepatocitos , Humanos , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Edición Génica/métodos , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Polimorfismo de Nucleótido Simple , Modelos BiológicosRESUMEN
Recent advances have made modeling human small intestines in vitro possible, but it remains a challenge to recapitulate fully their structural and functional characteristics. We suspected interstitial flow within the intestine, powered by circulating blood plasma during embryonic organogenesis, to be a vital factor. We aimed to construct an in vivo-like multilayered small intestinal tissue by incorporating interstitial flow into the system and, in turn, developed the micro-small intestine system by differentiating definitive endoderm and mesoderm cells from human pluripotent stem cells simultaneously on a microfluidic device capable of replicating interstitial flow. This approach enhanced cell maturation and led to the development of a three-dimensional small intestine-like tissue with villi-like epithelium and an aligned mesenchymal layer. Our micro-small intestine system not only overcomes the limitations of conventional intestine models but also offers a unique opportunity to gain insights into the detailed mechanisms underlying intestinal tissue development.
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In the evolving field of drug discovery and development, multiorgans-on-a-chip and microphysiological systems are gaining popularity owing to their ability to emulate in vivo biological environments. Among the various gut-liver-on-a-chip systems for studying oral drug absorption, the chip developed in this study stands out with two distinct features: incorporation of perfluoropolyether (PFPE) to effectively mitigate drug sorption and a unique enterohepatic single-passage system, which simplifies the analysis of first-pass metabolism and oral bioavailability. By introducing a bolus drug injection into the liver compartment, hepatic extraction alone could be evaluated, further enhancing our estimation of intestinal availability. In a study on midazolam (MDZ), PFPE-based chips showed more than 20-times the appearance of intact MDZ in the liver compartment effluent compared to PDMS-based counterparts. Notably, saturation of hepatic metabolism at higher concentrations was confirmed by observations when the dose was reduced from 200 µM to 10 µM. This result was further emphasized when the metabolism was significantly inhibited by the coadministration of ketoconazole. Our chip, which is designed to minimize the dead volume between the gut and liver compartments, is adept at sensitively observing the saturation of metabolism and the effect of inhibitors. Using genome-edited CYP3A4/UGT1A1-expressing Caco-2 cells, the estimates for intestinal and hepatic availabilities were 0.96 and 0.82, respectively; these values are higher than the known human in vivo values. Although the metabolic activity in each compartment can be further improved, this gut-liver-on-a-chip can not only be used to evaluate oral bioavailability but also to carry out individual assessment of both intestinal and hepatic availability.
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Disponibilidad Biológica , Éteres , Fluorocarburos , Hígado , Hígado/metabolismo , Fluorocarburos/química , Fluorocarburos/farmacocinética , Fluorocarburos/metabolismo , Humanos , Administración Oral , Dispositivos Laboratorio en un Chip , Células CACO-2 , Citocromo P-450 CYP3A/metabolismo , AnimalesRESUMEN
Prader-Willi syndrome (PWS), which is a complex epigenetic disorder caused by the deficiency of paternally expressed genes in chromosome 15q11-q13, is associated with several psychiatric dimensions, including autism spectrum disorder. We have previously reported that iPS cells derived from PWS patients exhibited aberrant differentiation and transcriptomic dysregulation in differentiated neural stem cells (NSCs) and neurons. Here, we identified SLITRK1 as a downregulated gene in NSCs differentiated from PWS patient iPS cells by RNA sequencing analysis. Because SLITRK1 is involved in synaptogenesis, we focused on the synaptic formation and function of neurons differentiated from PWS patient iPS cells and NDN or MAGEL2 single gene defect mutant iPS cells. Although ßIII tubulin expression levels in all the neurons were comparable to the level of differentiation in the control, pre- and postsynaptic markers were significantly lower in PWS and mutant neurons than in control neurons. PSD-95 puncta along ßIII tubulin neurites were also decreased. Membrane potential responses were measured while exposed to high K+ stimulation. The neuronal excitabilities in PWS and mutant neurons showed significantly lower intensity than that of control neurons. These functional defects in PWS neurons may reflect phenotypes of neurodevelopmental disorders in PWS.
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Trastorno del Espectro Autista , Células-Madre Neurales , Síndrome de Prader-Willi , Humanos , Síndrome de Prader-Willi/genética , Tubulina (Proteína)/genética , Neuronas , Cromosomas Humanos Par 15 , Proteínas/genéticaRESUMEN
In nonclinical studies, models that can predict the metabolism of drug candidates by cytochrome P450 (CYP), including Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) are helpful. CYP3A4-overexpressing human cells have been used universally to evaluate whether CYP3A4 metabolizes drug-candidate compounds. However, CYP3A4-overexpressing human cell lines are problematic because their activity levels are lower than that of in vivo human CYP3A4. Heme plays a paramount role in CYP activity. The rate-limiting step in heme biosynthesis is the generation of 5-aminolevulinic acid (5-ALA). In this study, we examined whether treatment with 5-ALA to CYP3A4-POR-UGT1A1-CES2 knockin and CES1 knockout (genome-edited) Caco-2 cells enhances CYP3A4 activity. A 7-day 5-ALA treatment increased intracellular heme levels in genome-edited Caco-2 cells without cytotoxicity. Moreover, consistent with the increase in intracellular heme content, 5-ALA treatment increased CYP3A4 activity in genome-edited Caco-2 cells. The results of this research are expected to be applied to pharmacokinetic studies using CYP-overexpressing human cells containing CYP3A4.
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Ácido Aminolevulínico , Citocromo P-450 CYP3A , Humanos , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Células CACO-2 , Ácido Aminolevulínico/farmacología , Hemo , Sistema Enzimático del Citocromo P-450/metabolismoRESUMEN
Caco-2 cells are widely used as an in vitro intestinal model. However, the expression levels of the drug-metabolizing enzymes CYP3A4 and UGT1A1 are lower in these cells than in intestinal cells. Furthermore, the majority of prodrugs in use today are ester-containing, and carboxylesterase (CES) 1 and CES2 are among the enzymes that process the prodrugs into drugs. In the human small intestine, CES1 is hardly expressed while CES2 is highly expressed, but the CES expression pattern in Caco-2 cells is the opposite. In this study, we generated CYP3A4-POR-UGT1A1-CES2 knock-in (KI) and CES1 knock-out (KO) Caco-2 (genome-edited Caco-2) cells using a PITCh system. Genome-edited Caco-2 cells were shown to express functional CYP3A4, POR, UGT1A1 and CES2 while the expression of the CES1 protein was completely knocked out. We performed transport assays using temocapril. The Papp value of temocapril in genome-edited Caco-2 cells was higher than that in WT Caco-2 cells. Interestingly, the amount of temocaprilat on the apical side in genome-edited Caco-2 cells was lower than that in WT Caco-2 cells. These results suggest that genome-edited Caco-2 cells are more suitable than WT Caco-2 cells as a model for predicting intestinal drug absorption and metabolism.
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Carboxilesterasa , Profármacos , Humanos , Células CACO-2 , Carboxilesterasa/genética , Carboxilesterasa/metabolismo , Citocromo P-450 CYP3A/genética , Profármacos/metabolismoRESUMEN
SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.
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HepG2 cells are an inexpensive hepatocyte model that can be used for repeated experiments, but HepG2 cells do not express major cytochrome P450s (CYPs) and UDP glucuronosyltransferase family 1 member A1 (UGT1A1). In this study, we established CYP3A4-POR-UGT1A1-CYP1A2-CYP2C19-CYP2C9-CYP2D6 (CYPs-UGT1A1) knock-in (KI)-HepG2 cells using a PITCh system to evaluate whether they could be a new hepatocyte model for pharmaceutical studies. To evaluate whether CYPs-UGT1A1 KI-HepG2 cells express and function with CYPs and UGT1A1, gene expression levels of CYPs and UGT1A1 were analyzed by using real-time PCR, and metabolites of CYPs or UGT1A1 substrates were quantified by HPLC. The expression levels of CYPs and UGT1A1 in the CYPs-UGT1A1 KI-HepG2 cells were comparable to those in primary human hepatocytes (PHHs) cultured for 48 h. The CYPs and UGT1A1 activity levels in the CYPs-UGT1A1 KI-HepG2 cells were much higher than those in the wild-type (WT)-HepG2 cells. These results suggest that the CYPs-UGT1A1 KI-HepG2 cells expressed functional CYPs and UGT1A1. We also confirmed that the CYPs-UGT1A1 KI-HepG2 cells were more sensitive to drug-induced liver toxicity than the WT-HepG2 cells. CYPs-UGT1A1 KI-HepG2 cells could be used to predict drug metabolism and drug-induced liver toxicity, and they promise to be a helpful new hepatocyte model for drug discovery research.
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Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450 , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Descubrimiento de Drogas , Células Hep G2 , Hepatocitos/metabolismo , HumanosRESUMEN
The small intestine plays a critical role in the absorption and metabolism of orally administered drugs. Therefore, a model capable of evaluating drug absorption and metabolism in the small intestine would be useful for drug discovery. Patients with genotype UGT1A1*6 (exon 1, 211G > A) treated with the antineoplastic drug SN-38 have been reported to exhibit decreased glucuronide conjugation and increased incidence of intestinal toxicity and its severe side effects, including severe diarrhea. To ensure the safety of drugs, we must develop a drug metabolism and toxicity evaluation model which considers UGT1A1*6. In this study, we generated CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells for pharmaceutical research using a PITCh system. The CYP3A4·POR·UGT1A1 KI-Caco-2 cells were shown to express functional CYP3A4 and UGT1A1. The CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells were sensitive to SN-38-induced intestinal toxicity. We thus succeeded in generating CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells, which can be used in pharmaceutical research. We also developed an intestinal epithelial cell model of patients with UGT1A1*6 and showed that it was useful as a tool for drug discovery.
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Citocromo P-450 CYP3A/genética , Glucuronosiltransferasa/genética , Mucosa Intestinal/enzimología , Intestino Delgado/enzimología , Antineoplásicos/toxicidad , Células CACO-2/enzimología , Descubrimiento de Drogas/métodos , Genotipo , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Irinotecán/toxicidadRESUMEN
A liver-on-a-chip (liver-chip) is a microfluidic device carrying liver cells such as human hepatocytes. It is used to reproduce a part of liver function. Many microfluidic devices are composed of polydimethylsiloxane (PDMS), which is a type of silicone elastomer. PDMS is easy to process and suitable for cell observation, but its high hydrophobicity carries the risk of drug absorption. In this study, we evaluated drug absorption to the PDMS device and investigated the drug responsiveness of human hepatocytes cultured in the PDMS device (hepatocyte-chips). First, the absorption rates of 12 compounds to the PDMS device were measured. The absorption rates of midazolam, bufuralol, cyclosporine A, and verapamil were 92.9, 71.7, 71.4, and 99.6%, respectively, but the other compounds were poorly absorbed. Importantly, the absorption rate of the compounds was correlated with their octanol/water distribution coefficient (logâ¯D) values (R2 = 0.76). Next, hepatocyte-chips were used to examine the response to drugs, which are typically used to evaluate hepatic functions. Using the hepatocyte-chips, we could confirm the responsiveness of drugs including cytochrome P450 (CYP) inducers and farnesoid X receptor (FXR) ligands. We believe that our findings will contribute to drug discovery research using PDMS-based liver-chips.
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Dispositivos Laboratorio en un Chip , Investigación Farmacéutica , Dimetilpolisiloxanos , Hepatocitos , Humanos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
The intestinal mucosa plays an important role as an immune barrier due to its continual exposure to invading pathogens, including viruses. It is thus highly important to evaluate virus infection profiles in the intestinal mucosa for prevention of virus infection and development of antivirus medicines; however, only a few enterocyte lines are available as in vitro intestinal models for the evaluation of virus infection. In this study, we evaluated profiles of infection and innate immune responses following infection with a mammalian orthoreovirus (hereafter reovirus), which has often been used as a tractable model for studies of viral pathogenesis, in human iPS cell-derived small intestinal epithelial-like cell (hiPS-SIEC) monolayers and cells of a human colon adenocarcinoma cell line, Caco-2. The levels of reovirus infection were similar between hiPS-SIEC and Caco-2 cell monolayers, which are often used as an intestinal model, after apical and basolateral infection. In hiPS-SIEC monolayers, more efficient replication of the virus genome was observed following basolateral infection than apical infection, while apical infection resulted in higher levels of virus protein expression and progeny virus production than basolateral infection. Reovirus significantly induced innate immune responses, including expression of type I and III interferons (IFNs), in hiPS-SIEC monolayers more efficiently than Caco-2 cells. Higher levels of type I and III interferon (IFN) expression were found in hiPS-SIEC monolayers following apical infection than basolateral infection. These results suggested that hiPS-SIECs are a promising in vitro model for the evaluation of virus infection.
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Células Madre Pluripotentes Inducidas , Orthoreovirus de los Mamíferos , Orthoreovirus , Reoviridae , Virosis , Animales , Células CACO-2 , Humanos , Inmunidad Innata , Interferones , Mamíferos , Orthoreovirus de los Mamíferos/genéticaRESUMEN
Sarcopenia, an age-related reduction in skeletal muscle mass and strength, is mainly caused by chronic inflammation. Because mesenchymal stem cells (MSCs) have the capacity to both promote myogenic cell differentiation and suppress inflammation, they are a promising candidate for sarcopenia treatment. In this study, to achieve the long-term retention of MSCs in skeletal muscle, we prepared magnetized MSCs using magnetic anionic liposome/atelocollagen complexes that we had previously developed, and evaluated their retention efficiency and immunomodulatory effects in mouse inflamed skeletal muscle. Mouse MSCs were efficiently magnetized by incubation with magnetic anionic liposome/atelocollagen complexes for 30 min under a magnetic field. The magnetized MSCs differentiated normally into osteoblasts and adipocytes. Additionally, non-magnetized MSCs and magnetized MSCs increased IL-6 and inducible nitric oxide synthase mRNA expression and decreased TNF-α and IL-1ß mRNA expression in C2C12 mouse skeletal muscle myotubes through paracrine effects. Moreover, magnetized MSCs were significantly retained in cell culture plates and mouse skeletal muscle after their local injection in the presence of a magnetic field. Furthermore, magnetized MSCs significantly increased IL-6 and IL-10 mRNA expression and decreased TNF-α and IL-1ß mRNA expression in inflamed skeletal muscle. These results suggest that magnetized MSCs may be useful for effective sarcopenia treatment.
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Células Madre Mesenquimatosas , Animales , Diferenciación Celular , Inmunomodulación , Liposomas , Fenómenos Magnéticos , Ratones , Músculo EsqueléticoRESUMEN
Human induced pluripotent stem cell-derived intestinal epithelial cells (hiPSC-IECs) are expected to be utilized in regenerative medicine. To perform a safe transplantation without the risk of tumor formation, residual undifferentiated hiPSCs must be removed from hiPSC-IECs. In this study, we examined whether vinblastine (a multiple drug resistance 1 [MDR1] substrate) could remove residual undifferentiated hiPSCs in hiPSC-IECs and attempted to generate hiPSC-IECs applicable to transplantation medicine. We found that the expression levels of pluripotent markers were largely decreased and those of intestinal markers were increased by vinblastine treatment. The treatment of undifferentiated hiPSCs with vinblastine significantly decreased their viability. These results suggested that undifferentiated hiPSCs can be eliminated from hiPSC-IECs by vinblastine treatment. We hypothesized that MDR1-negative cells (such as undifferentiated hiPSCs) die upon vinblastine treatment because they are unable to excrete vinblastine. As expected, the cell viability of MDR1-knockout hiPSC-IECs was significantly decreased by vinblastine treatment. Furthermore, teratomas were formed by subcutaneous transplantation of hiPSC-IECs mixed with undifferentiated hiPSCs into mice, but they were not observed when the transplanted cells were pre-treated with vinblastine. Vinblastine-treated hiPSC-IECs would be an effective cell source for safe regenerative medicine.
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Oxidative stress has been implicated in a variety of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Astrocytes play a significant role in maintaining survival of neurons by supplying antioxidants such as glutathione (GSH) to neurons. Recently, we found that noradrenaline increased the intracellular GSH concentration in astrocytes via ß3 -adrenoceptor stimulation. These observations suggest that noradrenaline protects neurons from oxidative stress-induced death by increasing the supply of GSH from astrocytes to neurons via the stimulation of ß3 -adrenoceptor in astrocytes. In the present study, we examined the protective effect of noradrenaline against H2 O2 -induced neurotoxicity using two different mixed cultures: the mixed culture of human astrocytoma U-251 MG cells and human neuroblastoma SH-SY5Y cells, and the mouse primary cerebrum mixed culture of neurons and astrocytes. H2 O2 -induced neuronal cell death was significantly attenuated by pretreatment with noradrenaline in both mixed cultures but not in single culture of SH-SY5Y cells or in mouse cerebrum neuron-rich culture. The neuroprotective effect of noradrenaline was inhibited by SR59230A, a selective ß3 -adrenoceptor antagonist, and CL316243, a selective ß3 -adrenoceptor agonist, mimicked the neuroprotective effect of noradrenaline. DL-buthionine-[S,R]-sulfoximine, a GSH synthesis inhibitor, negated the neuroprotective effect of noradrenaline in both mixed cultures. MK571, which inhibits the export of GSH from astrocytes mediated by multidrug resistance-associated protein 1, also prevented the neuroprotective effect of noradrenaline. These results suggest that noradrenaline protects neurons against H2 O2 -induced death by increasing the supply of GSH from astrocytes via ß3 -adrenoceptor stimulation.
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Astrocitos/efectos de los fármacos , Glutatión/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Norepinefrina/farmacología , Receptores Adrenérgicos beta 3/fisiología , Agonistas de Receptores Adrenérgicos beta 3/farmacología , Antagonistas de Receptores Adrenérgicos beta 3/farmacología , Animales , Astrocitos/metabolismo , Astrocitoma , Encéfalo/citología , Butionina Sulfoximina/farmacología , Línea Celular Tumoral , Técnicas de Cocultivo , Dioxoles/farmacología , Humanos , Peróxido de Hidrógeno/toxicidad , Ratones , Ratones Endogámicos C57BL , Neuroblastoma , Estrés Oxidativo , Propanolaminas/farmacología , Propionatos/farmacología , Quinolinas/farmacologíaRESUMEN
Mesenchymal stem cells (MSCs) are capable of repairing skeletal muscle via paracrine mechanisms. This regenerative effect of MSCs on skeletal muscle is based on promoting the proliferation and differentiation of myogenic cells and inhibiting the inflammatory response of immune cells. However, it is unclear whether MSCs affect the inflammatory response of skeletal muscle cells. In this study, we evaluated the paracrine effect of mouse MSCs on the inflammatory response of lipopolysaccharide (LPS)-stimulated C2C12 mouse myoblasts. Interleukin (IL)-6 production from LPS-stimulated C2C12 cells was significantly increased by coculture with MSCs or culture in conditioned medium of MSCs. This increased IL-6 production from C2C12 cells was not significantly suppressed by inhibiting mitogen-activated protein kinase pathways, but it was significantly suppressed by pretreatment with nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) inhibitors. In addition, IL-6 and inducible nitric oxide synthase (iNOS) mRNA expression was increased significantly in C2C12 cells cocultured with MSCs, while tumor necrosis factor (TNF)-α and IL-1ß mRNA expression was decreased. Furthermore, conditioned medium of C2C12 cells cocultured with MSCs exerted remarkable anti-inflammatory effects on LPS-stimulated mouse macrophages.
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Sistema de Señalización de MAP Quinasas/inmunología , Células Madre Mesenquimatosas/metabolismo , Mioblastos Esqueléticos/inmunología , Comunicación Paracrina/inmunología , Animales , Diferenciación Celular/inmunología , Línea Celular , Proliferación Celular , Técnicas de Cocultivo , Medios de Cultivo Condicionados/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/inmunología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Mioblastos Esqueléticos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Multiple drug resistance 1 (MDR1) is highly expressed in various organs, including the liver, small intestine, and blood-brain barrier (BBB). Because MDR1 plays important roles in the excretion of many drugs, it is necessary to evaluate whether drug candidates are potential substrates of MDR1. Recently, many researchers have shown that human induced pluripotent stem (iPS) cell-derived differentiated cells such as hepatocytes and enterocytes can be applied for pharmacokinetic testing. Here, we attempted to generate MDR1-knockout (KO) iPS cell lines using genome editing technology. The correctly targeted human iPS cell lines were successfully obtained. The expression levels of pluripotent markers in human iPS cells were not changed by MDR1 knockout. The gene expression levels of hepatic markers in MDR1-KO iPS-derived hepatocyte-like cells were higher than those in undifferentiated MDR1-KO iPS cells, suggesting that MDR1-KO iPS cells have hepatic differentiation capacity. In addition, MDR1 expression levels were hardly detected in MDR1-KO iPS cell-derived hepatocyte-like cells. We thus succeeded in establishing MDR1-KO iPS cell lines that could be utilized for pharmacokinetic testing.
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Células Madre Pluripotentes Inducidas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Diferenciación Celular , Edición Génica , Humanos , Células Madre Pluripotentes Inducidas/citologíaRESUMEN
Because many peptide and peptide-mimetic drugs are substrates of peptide transporter 1, it is important to evaluate the peptide transporter 1-mediated intestinal absorption of drug candidates in the early phase of drug development. Although intestinal cell lines treated with inhibitors of peptide transporter 1 are widely used to examine whether drug candidates are substrates for peptide transporter 1, these inhibitors are not sufficiently specific for peptide transporter 1. In this study, to generate a more precise evaluation model, we established peptide transporter 1-knockout induced pluripotent stem cells (iPSCs) by using a CRISPR-Cas9 system and differentiated the cells into intestinal epithelial-like cells. The permeability value and uptake capacity of glycylsarcosine (substrate of peptide transporter 1) in peptide transporter 1-knockout intestinal epithelial-like cells were significantly lower than those in wild-type intestinal epithelial-like cells, suggesting that peptide transporter 1 was successfully depleted in the epithelial cells. Taken together, our model can be useful in the development of peptide and peptide-mimetic drugs.
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BACKGROUND & AIMS: To develop an effective and safe orally administered drug, it is important to predict its intestinal absorption rate, intestinal first-pass effect, and drug-drug interactions of orally administered drugs. However, there is no existing model to comprehensively predict the intestinal pharmacokinetics and drug-response of orally administered drugs. In this study, we attempted to generate homogenous and functional intestinal epithelial cells from human induced pluripotent stem (iPS) cells for pharmaceutical research. METHODS: We generated almost-homogenous Villin- and zonula occludens-1 (ZO1)-positive intestinal epithelial cells by caudal-related homeobox transcription factor 2 (CDX2) transduction into human iPS cell-derived intestinal progenitor cells. RESULTS: The drug absorption rates in human iPS cell-derived intestinal epithelial cell monolayers (iPS-IECM) were highly correlated with those in humans (R2=0.91). The expression levels of cytochrome P450 (CYP) 3A4, a dominant drug-metabolizing enzyme in the small intestine, in human iPS-IECM were similar to those in human small intestine in vivo. In addition, intestinal availability in human iPS-IECM (the fraction passing the gut wall: Fg=0.73) was more similar to that in the human small intestine in vivo (Fg=0.57) than to that in Caco-2 cells (Fg=0.99), a human colorectal adenocarcinoma cell line. Moreover, the drug-drug interaction and drug-food interaction could be observed by using our human iPS-IECM in the presence of an inducer and inhibitor of CYP3A4, i.e., rifampicin and grape fruit juice, respectively. CONCLUSION: Taking these results together, we succeeded in generating the human iPS-IECM that can be applied to various intestinal pharmacokinetics and drug-response tests of orally administered drugs.
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Factor de Transcripción CDX2/genética , Células Madre Pluripotentes Inducidas/citología , Intestinos/citología , Transducción Genética/métodos , Factor de Transcripción CDX2/metabolismo , Células CACO-2 , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Interacciones Alimento-Droga , Jugos de Frutas y Vegetales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Absorción Intestinal , Rifampin/farmacocinéticaRESUMEN
The small intestine plays an important role in the absorption and metabolism of oral drugs. In the current evaluation system, it is difficult to predict the precise absorption and metabolism of oral drugs. In this study, we generated small intestinal epithelial-like cells from human induced pluripotent stem cells (hiPS-SIECs), which could be applied to drug absorption and metabolism studies. The small intestinal epithelial-like cells were efficiently generated from human induced pluripotent stem cell by treatment with WNT3A, R-spondin 3, Noggin, EGF, IGF-1, SB202190, and dexamethasone. The gene expression levels of small intestinal epithelial cell (SIEC) markers were similar between the hiPS-SIECs and human adult small intestine. Importantly, the gene expression levels of colonic epithelial cell markers in the hiPS-SIECs were much lower than those in human adult colon. The hiPS-SIECs generated by our protocol exerted various SIEC functions. In conclusion, the hiPS-SIECs can be utilized for evaluation of drug absorption and metabolism.
Asunto(s)
Células Epiteliales/metabolismo , Células Madre Pluripotentes Inducidas/citología , Absorción Intestinal , Intestino Delgado/citología , Preparaciones Farmacéuticas/metabolismo , Animales , Biomarcadores/metabolismo , Células CACO-2 , Proteínas Portadoras/farmacología , Diferenciación Celular/efectos de los fármacos , Dexametasona/farmacología , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Imidazoles/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Absorción Intestinal/efectos de los fármacos , Maleimidas/farmacología , Ratones , Piridinas/farmacología , Trombospondinas/farmacología , Proteína Wnt3A/farmacologíaRESUMEN
Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test.