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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.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Intestinos , Intestino Delgado/metabolismo , Enterócitos/metabolismo , Diferenciação CelularRESUMO
We present a systematic review of the methods developed for the synthesis of the aromathecin family of compounds (benz[6,7]indolizino[1,2-b]quinolin-11(13H)-ones) and their derivatives. These methods can be broadly classified into four categories based on the construction of pentacyclic structures: Category 1: by constructing a pyridone moiety (D-ring) on the pyrroloquinoline ring (A/B/C-ring), Category 2: by constructing a pyridine moiety (B-ring) on the pyrroloisoquinolone ring (C/D/E-ring), Category 3: by constructing an indolizidinone moiety (C/D-ring) in a tandem reaction, and Category 4: by constructing a pyrrolidine moiety (C-ring) on the isoquinolone ring (D/E-ring).
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Ca2+-dependent activator protein for secretion 2 (CAPS2) regulates dense-core vesicle (DCV) exocytosis to facilitate peptidergic and catecholaminergic transmitter release. CAPS2 deficiency in mice has mild neuronal effects but markedly impairs social behavior. Rare de novo Caps2 alterations also occur in autism spectrum disorder, although whether CAPS2-mediated release influences social behavior remains unclear. Here, we demonstrate that CAPS2 is associated with DCV exocytosis-mediated release of the social interaction modulatory peptide oxytocin (OXT). CAPS2 is expressed in hypothalamic OXT neurons and localizes to OXT nerve projection and OXT release sites, such as the pituitary. Caps2 KO mice exhibited reduced plasma albeit increased hypothalamic and pituitary OXT levels, indicating insufficient release. OXT neuron-specific Caps2 conditional KO supported CAPS2 function in pituitary OXT release, also affording impaired social interaction and recognition behavior that could be ameliorated by exogenous OXT administered intranasally. Thus, CAPS2 appears critical for OXT release, thereby being associated with social behavior.SIGNIFICANCE STATEMENT The role of the neuropeptide oxytocin in enhancing social interaction and social bonding behavior has attracted considerable public and neuroscientific attention. A central issue in oxytocin biology concerns how oxytocin release is regulated. Our study provides an important insight into the understanding of oxytocin-dependent social behavior from the perspective of the CAPS2-regulated release mechanism.
Assuntos
Comportamento Animal/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Exocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Ocitocina/metabolismo , Comportamento Social , Animais , Hipotálamo/metabolismo , Camundongos , Camundongos Knockout , Vesículas Secretórias/metabolismoRESUMO
OBJECTIVE AND DESIGN: The primary component in gut mucus is mucin 2 (MUC2) secreted by goblet cells. Fluctuations in MUC2 expression are considered a useful indicator for evaluating mucosal damage and protective effect of various agents using animal studies. However, there are few in vitro studies evaluating mucosal damage using MUC2 as the indicator. Hence, we attempted to establish a novel in vitro model with MUC2 as the indicator for evaluating drug-induced mucosal damage and protective effect using enterocytes derived from human iPS cells. METHODS: Compounds were added into enterocytes derived from human iPS cells, and MUC2 mRNA and protein expression levels were evaluated. Further, the effect of compounds on membrane permeability was investigated. RESULTS: Nonsteroidal anti-inflammatory drugs were found to decrease MUC2 mRNA expression in enterocytes, whereas mucosal protective agents increased mRNA levels. Changes in MUC2 protein expression were consistent with those of mRNA. Additionally, our results indicated that indomethacin caused mucosal damage, affecting membrane permeability of the drug. Moreover, we observed protective effect of rebamipide against the indomethacin-induced permeability increase. CONCLUSIONS: The developed model could facilitate evaluating drug-induced mucosal damage and protective effects of various agents and could impact drug development studies regarding pharmacological efficacy and safety.
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Alanina/análogos & derivados , Anti-Inflamatórios não Esteroides/toxicidade , Antiulcerosos/farmacologia , Enterócitos/efeitos dos fármacos , Indometacina/toxicidade , Mucina-2/metabolismo , Quinolonas/farmacologia , Alanina/farmacologia , Alternativas aos Testes com Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Enterócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mucina-2/genéticaRESUMO
The hypervalent iodine/HF reagent consisting of PhIO and HF·py was found to be effective for fluorination of functionalized aromatic olefins bearing synthetically important carbonyl and hydroxyl groups. Fluorination of 1,3-diphenyl-2-propen-1-one with PhIO/HF·py reagent in CH2Cl2 at room temperature gave 3,3-difluoro-1,2-diphenyl-1-propanone in high yield. Other α-aryl-α,ß-unsaturated ketones underwent the fluorination to yield aryl 2,2-difluoroethyl ketone derivatives in good to high yields. Catalytic fluorination of α-aryl-α,ß-unsaturated ketones using a p-TolI/HF·py/mCPBA reagent system also worked well. Moreover, the fluorination of cinnamyl alcohol derivatives by PhIO/HF·py reagent proceeded smoothly to afford 2-aryl-3,3-difluoro-1-propanols in moderate yields.
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The reaction of alkynes with PhIO and Py·HF followed by treatment with BF3·OEt2 gave ß-fluorovinyliodonium tetrafluoroborates in good to high yields. More conveniently, the reaction using PhI and Py·HF in the presence of m-CPBA also afforded ß-fluorovinyliodonium tetrafluoroborates in good yields. These methods have the advantages that ß-fluorovinyliodonium salts can be prepared without ArIF2.
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We demonstrated the ruthenium-catalyzed allylic amination of 2,3,3-trifluoroallylic carbonates with several types of amines. The reactions proceeded with several types of amines, and succeeded in obtaining polyfluorinated terminal alkenes possessing branched allylic amines as a single regioisomer.
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We demonstrated the kinetic resolution of racemic and branched monosubstituted allylic acetates by a ruthenium-catalyzed regioselective allylic etherification. The reaction was effectively catalyzed by the chiral ruthenium catalyst, which was generated by [RuCl2(p-cymene)]2 and (S,S)-iPr-pybox and a catalytic amount of TFA, and both the allylic etherification product and recovered allylic acetate were obtained as an enantiomerically enriched form with up to a 103 s value.
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Whole-genome sequencing (WGS) studies of autism spectrum disorder (ASD) have demonstrated the roles of rare promoter de novo variants (DNVs). However, most promoter DNVs in ASD are not located immediately upstream of known ASD genes. In this study analyzing WGS data of 5,044 ASD probands, 4,095 unaffected siblings, and their parents, we show that promoter DNVs within topologically associating domains (TADs) containing ASD genes are significantly and specifically associated with ASD. An analysis considering TADs as functional units identified specific TADs enriched for promoter DNVs in ASD and indicated that common variants in these regions also confer ASD heritability. Experimental validation using human induced pluripotent stem cells (iPSCs) showed that likely deleterious promoter DNVs in ASD can influence multiple genes within the same TAD, resulting in overall dysregulation of ASD-associated genes. These results highlight the importance of TADs and gene-regulatory mechanisms in better understanding the genetic architecture of ASD.
Assuntos
Transtorno do Espectro Autista , Células-Tronco Pluripotentes Induzidas , Humanos , Transtorno do Espectro Autista/genética , Predisposição Genética para Doença/genética , Regulação da Expressão Gênica , Sequenciamento Completo do GenomaRESUMO
Human induced pluripotent stem (iPS) cell-derived intestinal organoids have low invasiveness; however, the current differentiation method does not reflect the crypt-villus-like structure due to structural immaturity. Here, we generated budding-like organoids that formed epithelial tissue-like structures and had the characteristics of the mature small intestine from human iPS cells. They showed a high expression of drug transporters and induced the expression of cytochrome P450 3A4 and P-glycoprotein. When treated with tumor necrosis factor-α and/or transforming growth factor-ß, the budding-like organoids replicated the pathogenesis of mucosal damage or intestinal fibrosis. Upon dissociation and seeding on cell culture inserts, the organoids retained intestinal characteristics, forming polarized intestinal folds with approximately 400 Ωâ¯×â¯cm2 transepithelial electrical resistance. This novel method has great potential for disease modeling and drug screening applications.
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Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Humanos , Mucosa Intestinal , Intestinos , OrganoidesRESUMO
The small intestine plays an important role in the pharmacokinetics of orally administered drugs due to the presence of drug transporters and drug-metabolizing enzymes. However, few appropriate methods exist to investigate intestinal pharmacokinetics. Induced pluripotent stem (iPS) cells can form various types of cells and represent a potentially useful tool for drug discovery. We previously reported that differentiated enterocytes from human iPS cells are useful for pharmacokinetic studies; however, the process is time and resource intensive. Here, we established a new two-dimensional culture method for maintaining human iPS-cell-derived intestinal stem cells (ISCs) with differentiation potency and evaluated their ability to differentiate into enterocytes exhibiting appropriate pharmacokinetic function. The culture method used several factors to activate signalling pathways required for maintaining stemness, followed by differentiation into enterocytes. Functional evaluation was carried out to verify epithelial-marker expression and inducibility and activity of metabolic enzymes and transporters. Our results confirmed the establishment of an ISC culture method for maintaining stemness and verified that the differentiated enterocytes from the maintained ISCs demonstrated proper pharmacokinetic function. Thus, our findings describe a time- and cost-effective approach that can be used as a general evaluation tool for evaluating intestinal pharmacokinetics.
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Diferenciação Celular , Técnicas de Cultura , Enterócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco/citologia , Biomarcadores , Meios de Cultura , Enterócitos/metabolismo , Imunofluorescência , Expressão Gênica , Humanos , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Mensageiro/genética , Células-Tronco/metabolismoRESUMO
Mouse line BTBR T+ Iptr3 tf /J (hereafter referred as to BTBR/J) is a mouse strain that shows lower sociability compared to the C57BL/6J mouse strain (B6) and thus is often utilized as a model for autism spectrum disorder (ASD). In this study, we utilized another subline, BTBRTF/ArtRbrc (hereafter referred as to BTBR/R), and analyzed the associated brain transcriptome compared to B6 mice using microarray analysis, quantitative RT-PCR analysis, various bioinformatics analyses, and in situ hybridization. We focused on the cerebral cortex and the striatum, both of which are thought to be brain circuits associated with ASD symptoms. The transcriptome profiling identified 1,280 differentially expressed genes (DEGs; 974 downregulated and 306 upregulated genes, including 498 non-coding RNAs [ncRNAs]) in BTBR/R mice compared to B6 mice. Among these DEGs, 53 genes were consistent with ASD-related genes already established. Gene Ontology (GO) enrichment analysis highlighted 78 annotations (GO terms) including DNA/chromatin regulation, transcriptional/translational regulation, intercellular signaling, metabolism, immune signaling, and neurotransmitter/synaptic transmission-related terms. RNA interaction analysis revealed novel RNA-RNA networks, including 227 ASD-related genes. Weighted correlation network analysis highlighted 10 enriched modules including DNA/chromatin regulation, neurotransmitter/synaptic transmission, and transcriptional/translational regulation. Finally, the behavioral analyses showed that, compared to B6 mice, BTBR/R mice have mild but significant deficits in social novelty recognition and repetitive behavior. In addition, the BTBR/R data were comprehensively compared with those reported in the previous studies of human subjects with ASD as well as ASD animal models, including BTBR/J mice. Our results allow us to propose potentially important genes, ncRNAs, and RNA interactions. Analysis of the altered brain transcriptome data of the BTBR/R and BTBR/J sublines can contribute to the understanding of the genetic underpinnings of autism susceptibility.
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Herein, we evaluated CYPs and their nuclear receptor mRNA induction by exposure to typical inducers, omeprazole, rifampicin, and phenobarbital in cynomolgus monkey hepatocytes. Six freshly-isolated hepatocytes and 6 cryopreserved hepatocytes from cynomolgus monkey liver were prepared for a 14-day monolayer culture, 28-day co-culture with feeder cells, and 28-day 3D spheroid culture with feeder cells. Omeprazole and rifampicin respectively induced CYP1A1 and CYP3A8 mRNAs, while phenobarbital induced CYP2C43, CYP2C75, and CYP3A8, and slightly induced CYP2B6. The nuclear receptors AHR, PXR, and CAR mRNA levels, which were activated by omeprazole, rifampicin, and phenobarbital, respectively, tended to decrease via exposure to inducers despite the increase in CYP mRNA levels. These trends were similar for all three culture methods. No evident difference was observed in CYP mRNA induction between fresh and cryopreserved hepatocytes. Based on mRNA levels, the co-culture and 3D spheroid culture methods are more reasonable than monolayer culture for CYP evaluation, because the use of feeder cells can reduce the number of hepatocytes, improve the cell adhesion, and maintain the mRNA expression levels. In addition, co-culture method is more cost-effective, as common culture plates can be used.
Assuntos
Indutores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/efeitos dos fármacos , Omeprazol/farmacologia , Fenobarbital/farmacologia , RNA Mensageiro/metabolismo , Rifampina/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Feminino , Hepatócitos/metabolismo , Fígado/citologia , Macaca fascicularis , Masculino , RNA Mensageiro/genéticaRESUMO
In this study, freshly isolated and cryopreserved cynomolgus monkey hepatocytes were seeded on Cell-able® plates with feeder cells to form spheroids and were cultured for 28 days. As a control, hepatocytes were also cultured with or without feeder cells on collagen-coated plates. We verified the mRNA expression levels of drug-metabolizing enzyme-related genes and the leakage of enzymes (AST, ALT, LDH, and γ-GTP) as indicators of cell survival. As a result, the patterns of target mRNA expression in fresh and cryopreserved hepatocytes were very similar during the culture period between culture methods. mRNA expression levels were highly maintained at day 28 using the 3D spheroid and co-culture methods, demonstrating that these methods are useful for maintenance of liver function. Leakage of AST and ALT was higher at day 3 but decreased at day 14. LDH was not detected, suggesting that the cell viability was also maintained during the culture period. Furthermore, the functional differences between fresh and cryopreserved hepatocytes were not clearly detected. The co-culture method was useful for long-term culture not requiring 3D structure, and the 3D spheroid culture method was effective as well. With these techniques, cynomolgus monkey hepatocytes are expected to exhibit smaller individual differences and high reproducibility.
Assuntos
Alanina Transaminase/genética , Aspartato Aminotransferases/genética , Criopreservação , L-Lactato Desidrogenase/genética , RNA Mensageiro/genética , gama-Glutamiltransferase/genética , Células 3T3 , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Sobrevivência Celular , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , L-Lactato Desidrogenase/metabolismo , Macaca fascicularis , Masculino , Camundongos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , gama-Glutamiltransferase/metabolismoRESUMO
Engulfment and cell motility (ELMO) proteins bind to Dock180, a guanine nucleotide exchange factor (GEF) of the Rac family, and regulate GEF activity. The resultant ELMO/Dock180/Rac module regulates cytoskeletal reorganization responsible for the engulfment of apoptotic cells, cell migration, and neurite extension. The expression and function of Elmo family proteins in the nervous system, however, are not yet fully understood. Here, we characterize the comparative gene expression profiles of three Elmo family members (Elmo1, Elmo2, and Elmo3) in the brain of C57BL/6J mice, a widely used inbred strain, together with reeler mutant mice to understand gene expression in normal laminated brain areas compared with abnormal areas. Although all three Elmo genes showed widespread mRNA expression over various mouse tissues tested, Elmo1 and Elmo2 were the major types expressed in the brain, and three Elmo genes were up-regulated between the first postnatal week (infant stage) and the third postnatal week (juvenile, weaning stage). In addition, the mRNAs of Elmo genes showed distinct distribution patterns in various brain areas and cell-types; such as neurons including inhibitory interneurons as well as some non-neuronal cells. In the cerebral cortex, the three Elmo genes were widely expressed over many cortical regions, but the predominant areas of Elmo1 and Elmo2 expression tended to be distributed unevenly in the deep (a lower part of the VI) and superficial (II/III) layers, respectively, which also changed depending on the cortical areas and postnatal stages. In the dentate gyrus of the hippocampus, Elmo2 was expressed in dentate granule cells more in the mature stage rather than the immature-differentiating stage. In the thalamus, Elmo1 but not the other members was highly expressed in many nuclei. In the medial habenula, Elmo2 and Elmo3 were expressed at intermediate levels. In the cerebellar cortex, Elmo1 and Elmo2 were expressed in differentiating-mature granule cells and mature granule cells, respectively. In the Purkinje cell layer, Elmo1 and Elmo2 were expressed in Purkinje cells and Bergmann glia, respectively. Disturbed cellular distributions and laminar structures caused by the reeler mutation did not severely change expression in these cell types despite the disturbed cellular distributions and laminar structures, including those of the cerebrum, hippocampus, and cerebellum. Taken together, these results suggested that these three Elmo family members share their functional roles in various brain regions during prenatal-postnatal development.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica/métodos , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Proteína Reelina , Serina Endopeptidases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
The ruthenium-catalyzed regioselective allylic amination of tertiary allylic acetates with several types of amines has been accomplished. The reaction was effectively catalyzed by Cp*RuCl2/5,5'-dimethyl-2,2'-bipyridine or its related ruthenium catalyst systems, and α,α-disubstituted allylic amines were formed as a single regioisomer in moderate to high yields.
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Hippocampal adult neurogenesis is observed in the subgranular zone of the dentate gyrus (DG), and is associated with hippocampal memory formation and several psychiatric disorders including autism spectrum disorder (ASD). Calcium-dependent activator protein for secretion 2 (CAPS2) is a candidate gene related to ASD, and is highly expressed in the hippocampal DG region, with Caps2 knockout (KO) mice exhibiting ASD-like behavior. Accordingly, CAPS2 is potentially associated with hippocampal adult neurogenesis, the relationship between CAPS2 and adult neurogenesis has not yet been investigated. Here, we determined whether deficit of the Caps2 gene affects hippocampal adult neurogenesis and maturation of newborn neurons. To induce adult neurogenesis, we used the environmental enrichment (EE) condition. Both wild-type (WT) and Caps2 KO mice were housed in control or EE conditions for 3 or 14days. Hippocampal levels of brain-derived neurotrophic factor (BDNF) can be used as a physiological EE conditioned marker, and were increased at 14days in the EE condition in both WT and KO mice. Newborn cells during control and EE conditions were labeled by BrdU, and the labeled cells co-immunostained with the immature and mature neuron markers, calretinin (CR) and NeuN. The ratio of CR/BrdU and NeuN/BrdU double positive cells to all of BrdU positive cells were significantly increased in WT mice housed in the EE condition for 14days compared with the control condition. Whereas KO mice in the EE condition showed no significant increase of newborn neurons. These findings suggest that CAPS2 deficiency strongly impairs hippocampal adult neurogenesis and maturation of newborn neurons.