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Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens, such as Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear translocation in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-ß family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear translocation. To understand the importance of core protein nuclear translocation, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These results suggest that IPO7-mediated nuclear translocation of core proteins is involved in the release of infectious virus particles of flaviviruses.
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Transporte Ativo do Núcleo Celular , Núcleo Celular , Flavivirus , Humanos , Flavivirus/metabolismo , Flavivirus/fisiologia , Animais , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Replicação Viral/fisiologia , Proteínas do Core Viral/metabolismo , Proteínas do Core Viral/genética , Carioferinas/metabolismo , Carioferinas/genética , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Chlorocebus aethiops , Células HEK293RESUMO
Incomplete Freund's adjuvant (IFA) has been used for many years to induce autoimmune diseases in animal models, including experimental autoimmune encephalitis and collagen-induced arthritis. However, it remains unclear why it is necessary to emulsify autoantigen and heat-killed Mycobacterium tuberculosis (HKMtb) with IFA to induce experimental autoimmune diseases. Here, we found that immunization with self-antigen and HKMtb was insufficient to induce autoimmune diseases in mice. Furthermore, IFA or one of its components, mineral oil, but not mannide monooleate, was required for the development of experimental autoimmune disease. Immunization with autoantigen and HKMtb emulsified in mineral oil facilitated innate immune activation and promoted the differentiation of pathogenic CD4+ T cells, followed by their accumulation in neuronal tissues. Several water-soluble hydrocarbon compounds were identified in mineral oil. Of these, immunization with HKMtb and autoantigen emulsified with the same amount of hexadecane or tridecylcyclohexane as mineral oil induced the development of experimental autoimmune encephalitis. In contrast, immunization with HKMtb and autoantigen emulsified with tridecylcyclohexane, but not hexadecane, at doses equivalent to those found in mineral oil, resulted in neuronal dysfunction. These data indicate that tridecylcyclohexane in mineral oil is a critical component in the induction of experimental autoimmune disease.
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Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.
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Neoplasias Hepáticas , Proteínas R-SNARE , Sinaptotagmina I , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Transporte Proteico , Proteínas R-SNARE/metabolismo , Sinaptotagmina I/metabolismo , Microambiente TumoralRESUMO
The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.
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MAP Quinases Reguladas por Sinal Extracelular , Neoplasias Pancreáticas , Humanos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosforilação , Transdução de Sinais , Neoplasias Pancreáticas/tratamento farmacológico , Apoptose , Sistema de Sinalização das MAP Quinases , Neoplasias PancreáticasRESUMO
BACKGROUND: Alcohol is a major abused drug worldwide that contributes substantially to health and social problems. These problems result from acute alcohol overuse as well as chronic use, leading to alcohol use disorder (AUD). A major goal of this field is to establish a treatment for alcohol abuse and dependence in patients with AUD. The central molecular mechanisms of acute alcohol actions have been extensively investigated in rodent models. AIMS: One of the central mechanisms that may be involved is glycogen synthase kinase-3ß (GSK-3ß) activity, a key enzyme involved in glycogen metabolism but which has crucial roles in numerous cellular processes. Although the exact mechanisms leading from acute alcohol actions to these chronic changes in GSK-3ß function are not yet clear, GSK-3ß nonetheless constitutes a potential therapeutic target for AUD by reducing its function using GSK-3ß inhibitors. This review is focused on the correlation between GSK-3ß activity and the degree of alcohol consumption. METHODS: Research articles regarding investigation of effect of GSK-3ß on alcohol consumption in rodents were searched on PubMed, Embase, and Scopus databases using keywords "glycogen synthase kinase," "alcohol (or ethanol)," "intake (or consumption)," and evaluated by changes in ratios of pGSK-3ßSer9/pGSK-3ß. RESULTS: In animal experiments, GSK-3ß activity decreases in the brain under forced and voluntary alcohol consumption while GSK-3ß activity increases under alcohol-seeking behavior. CONCLUSIONS: Several pieces of evidence suggest that alterations in GSK-3ß function are important mediators of chronic ethanol actions, including those related to alcohol dependence and the adverse effects of chronic ethanol exposure.
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Encéfalo , Etanol , Animais , Humanos , Glicogênio Sintase Quinase 3 beta/metabolismo , Etanol/efeitos adversos , Encéfalo/metabolismo , Consumo de Bebidas Alcoólicas/metabolismo , FosforilaçãoRESUMO
Human induced pluripotent stem (hiPS) cells have demonstrated promising potential in regenerative medical therapeutics. After successful clinical trials, the demand for hiPS cells has steadily increased. Therefore, the optimization of hiPS cell freezing processes for storage and transportation is essential. Here, we presented a computer-aided exploration of multiobjective optimal temperature profiles in slow freezing for hiPS cells. This study was based on a model that calculates cell survival rates after thawing, and the model was extended to evaluate cell potentials until 24 h after seeding. To estimate parameter values for this extension, freezing experiments were performed using constant cooling rates. Using quality and productivity indicators, we evaluated 16,206 temperature profiles using our model, and a promising profile was obtained. Finally, an experimental investigation of the profile was undertaken, and the contribution of the temperature profile to both quality and productivity was confirmed.
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Sobrevivência Celular , Criopreservação , Congelamento , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Criopreservação/métodos , Temperatura , Simulação por ComputadorRESUMO
Cellular homeostasis is assumed to be regulated by the coordination of dynamic behaviors. Lack of efficient methods for synchronizing large quantities of cells makes studying cell culture strategies for bioprocess development challenging. Here, we demonstrate a novel application of botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, to synchronize behavior-driven mechanical memory in human induced pluripotent stem cell (hiPSC) cultures. Application of HA to hiPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration-and time-dependent manner. Interestingly, cytoskeleton rearrangement in cells with prolonged exposure to HA resulted in mechanical memory synchronization with Yes-associated protein, which increased pluripotent cell homogeneity. Synchronized hiPSCs have higher capability to differentiate into functional hepatocytes than unsynchronized hiPSCs, resulting in improved efficiency and robustness of hepatocyte differentiation. Thus, our strategy for cell behavior synchronization before differentiation induction provides an approach against the instability of differentiation of pluripotent cells.
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Clostridium botulinum , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular , Técnicas de Cultura de Células , HepatócitosRESUMO
Three-dimensional (3D) culture platforms have been explored to establish physiologically relevant cell culture environment and permit expansion scalability; however, little is known about the mechanisms underlying the regulation of pluripotency of human induced pluripotent stem cells (hiPSCs). This study elucidated epigenetic modifications contributing to pluripotency of hiPSCs in response to 3D culture. Unlike two-dimensional (2D) monolayer cultures, 3D cultured cells aggregated with each other to form ball-like aggregates. 2D cultured cells expressed elevated levels of Rac1 and RhoA; however, Rac1 level was significantly lower while RhoA level was persisted in 3D aggregates. Compared with 2D monolayers, the 3D aggregates also exhibited significantly lower myosin phosphorylation. Histone methylation analysis revealed remarkable H3K4me3 upregulation and H3K27me3 maintenance throughout the duration of 3D culture; in addition, we observed the existence of naïve pluripotency signatures in cells grown in 3D culture. These results demonstrated that hiPSCs adapted to 3D culture through alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and transcriptional expression of pluripotency-associated factors. These results may help design culture environments for stable and high-quality hiPSCs.
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Citoesqueleto de Actina/metabolismo , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Linhagem Celular , Epigênese Genética/genética , Código das Histonas/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas rac1 de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/biossínteseRESUMO
Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4-/- ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3-/- ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4-/- mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4-/- mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4-/- mice. A five-fold increase in the frequency of abnormalities in Kpna4-/- mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4-/- mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4-/- testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.
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Fertilidade/genética , Infertilidade Masculina/genética , Carioferinas/genética , Motilidade dos Espermatozoides/genética , Espermatozoides/anormalidades , alfa Carioferinas/genética , Reação Acrossômica/genética , Animais , Flagelos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatogênese/genética , Testículo/anormalidadesRESUMO
Fully realizing the enormous potential of stem cells requires developing efficient bioprocesses and optimizations founded in mechanobiological considerations. Here, we emphasize the importance of mechanotransduction as one of the governing principles of stem cell bioprocesses, underscoring the need to further explore the behavioral mechanisms involved in sensing mechanical cues and coordinating transcriptional responses. We identify the sources of intrinsic, extrinsic, and external noise in bioprocesses requiring further study, and discuss the criteria and indicators that may be used to assess and predict cell-to-cell variability resulting from environmental fluctuations. Specifically, we propose a conceptual framework to explain the impact of mechanical forces within the cellular environment, identify key cell state determinants in bioprocesses, and discuss downstream implementation challenges.
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Biofísica , Reatores Biológicos , Mecanotransdução Celular/fisiologia , Células-Tronco , Biotecnologia , Técnicas de Cultura de Células , Humanos , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Existing guidelines form no consensus for alopecia areata (AA) treatment due to the absence of a universal standard treatment and arbitrary selection of reference arms in randomized control trials (RCTs). The aim is to identify the best treatment and to rank treatments using systematic review and network meta-analysis. Data were extracted by the two investigators independently. Odds ratio (OR) of treatment success rate was pooled using the frequentist weighted least squares approach to random-model network meta-analysis. RCTs providing data of treatment success rate from PubMed, EMBASE, Web of Science, and manual search were included. About 54 RCTs consisting of 49 treatments and 3149 patients were included. Pentoxifylline plus topical corticosteroids had the highest treatment success rate compared with "no treatment," followed by pentoxifylline alone, topical calcipotriol plus narrowband ultraviolet radiation B phototherapy, topical calcipotriol, intralesional corticosteroids, systemic corticosteroids, minoxidil plus topical corticosteroids, topical bimatoprost, psoralen ultraviolet radiation A phototherapy, and tofacitinib. Even with the network meta-analysis, the best treatment because of independent loops and wide confidence intervals could not be identified. Treatment options above may be reasonable strategies, but further comparison is required.
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Alopecia em Áreas , Terapia Ultravioleta , Alopecia em Áreas/diagnóstico , Alopecia em Áreas/tratamento farmacológico , Humanos , Minoxidil , Metanálise em Rede , FototerapiaRESUMO
Adenine-thymine (AT)-rich interactive domain 5a (Arid5a) is an RNA-binding protein found in the cytoplasm and nucleus of normally growing cells. Although Arid5a is known to play an important role in immune regulation, whether and how Arid5a subcellular localization impacts immune regulation has remained unclear. In this study, we generated Arid5a transgenic (TG) mice to address this question. While ectopic Arid5a overexpression did not affect expression of inflammatory cytokines under unstimulated conditions, significantly higher levels of inflammatory cytokines, such as IL-6, were produced in response to lipopolysaccharide (LPS) stimulation. Consistent with this, TG mice were more sensitive to LPS treatment than wild-type mice. We also found that Arid5a is imported into the nucleus via a classical importin-α/ß1-mediated pathway. On stimulation, nuclear Arid5a levels were decreased, while there was a concomitant increase in cytoplasmic Arid5a. Arid5a is associated with up-frameshift protein 1, and its nuclear export is regulated by a nuclear export receptor, chromosomal region maintenance 1. Taken together, these data indicate that Arid5a is a dynamic protein that translocates to the cytoplasm from the nucleus so as to properly exert its dual function in mRNA stabilization and transcriptional regulation during inflammatory conditions.
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Núcleo Celular/metabolismo , Citocinas/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/fisiologia , Inflamação/imunologia , Ribonucleases/metabolismo , Fatores de Transcrição/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Feminino , Células HeLa , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Frações SubcelularesRESUMO
Invited for the cover of this issue is Hiroaki Ohno and co-workers at Kyoto University and National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN). The image depicts gold catalysis which promotes the domino reaction to push the switch for the diversity-directed total synthesis. Read the full text of the article at 10.1002/chem.202001950.
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The total synthesis of dictyodendrins A-F was achieved by using the gold(I)-catalyzed annulation of a conjugated diyne with N-Boc-pyrrole for direct construction of the pyrrolo[2,3-c]carbazole scaffold. Late-stage functionalization of the resulting pyrrolo[2,3-c]carbazole to introduce various substituents provided divergent access to dictyodendrins. Some dictyodendrin analogues exhibited inhibitory activities toward CDK2/CycA2 and GSK3.
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The creation of a blueprint for stem cell bioprocess development that it is easily readable and shareable among those involved in the construction of the bioprocess is a necessary step toward full-fledged bioprocess integration. The blueprint provides the culturing tools and methodologies, designed to highlight knowledge gaps within biological sciences and bioengineering. This review highlights a blueprint for stem cell bioprocessing development using a landscape architecture approach that can aid the development of culture technologies and tools that satisfy the demands for stem cell-derived products for use in clinical and industrial applications. This work is intended to provide insights to cell biologists, geneticists, bioengineers, and clinicians seeking knowledge outside of their field of expertise and fosters a leap from a reductionist approach to one, that is, globally integrated in stem cell bioprocessing.
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Bioengenharia , Reatores Biológicos , Técnicas de Cultura de Células , Células-Tronco , Diferenciação Celular , Células Cultivadas , Epigênese Genética , Humanos , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
BACKGROUND: Clostridium perfringens can cause various infections, including food poisoning, gas gangrene, cellulitis and fasciitis. C. perfringens septicemia is rare, but is a known cause of hemolysis by damaging red blood cell, and often proves rapidly fatal in emergency department (ED) situations. CASE PRESENTATION: A previously healthy 76-year-old man presented to the ED 8 h after onset of acute abdominal pain and diarrhea. Laboratory examination revealed a large discrepancy between the red blood cell count of 1.91 × 106/mm3 and the hemoglobin level of 10.3 g/dL, suggesting massive intravascular hemolysis. Computed tomography revealed liver abscesses with gas. During ED treatment, the state of the patient rapidly deteriorated and he entered cardiopulmonary arrest. Blood cultures finally identified C. perfringens. CONCLUSION: Intravascular hemolysis and red blood cell (RBC) / hemoglobin (Hb) discrepancy in the presence of infection should prompt ED physicians to consider C. perfringens septicemia and to act quickly to provide appropriate treatment.
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Infecções por Clostridium/diagnóstico , Clostridium perfringens/patogenicidade , Gangrena Gasosa/diagnóstico , Hemoglobinas/análise , Abscesso Hepático/microbiologia , Idoso , Bacteriemia/microbiologia , Hemocultura , Infecções por Clostridium/etiologia , Gangrena Gasosa/etiologia , Parada Cardíaca , Hemólise , Humanos , Abscesso Hepático/diagnóstico por imagem , Masculino , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Dedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets. RESULTS: During the first two passages (P0 - P2), the relative mRNA level of collagen type II decreased in all cultures, while that of collagen type I increased. Remarkably, the level of collagen type II was higher and aggrecan was retained in the chondrocytes, forming cell aggregates and showing some round-shaped cells with less production of stress fibers on the G5 surface compared to fibroblast-like chondrocytes with abundant stress fibers on the PS surface. The numbers of P2 chondrocytes on the G5 and PS surfaces were nearly the same and sufficient for construction of chondrocyte sheets using a temperature-responsive plate. Without a supporting material during cell sheet manipulation, chondrocyte sheets spontaneously detached and exhibited a honeycomb-like structure of stress fibers. Unlike the chondrocyte sheets constructed from cells on the PS surface, the chondrocyte sheets from cells on the G5 surface had higher chondrogenic activities, as evidenced by the high expression of chondrogenic markers and the low expression of dedifferentiation markers. CONCLUSIONS: The one-step process of cell expansion and maintenance of chondrogenic activity could be obtained using the G5 surface. Human chondrocyte sheets were successfully constructed with high chondrogenic activity. These findings may lead to an alternative cultivation technique for human chondrocytes that offers high clinical potential in autologous chondrocyte implantation.
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Técnicas de Cultura de Células/métodos , Condrócitos/citologia , Condrócitos/fisiologia , Dendrímeros/química , Idoso , Agrecanas/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Colágeno Tipo II/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Propriedades de SuperfícieRESUMO
PKN2, a member of the protein kinase N (PKN) family, has been suggested by in vitro culture cell experiments to bind to Rho/Rac GTPases and contributes to cell-cell contact and cell migration. To unravel the in vivo physiological function of PKN2, we targeted the PKN2 gene. Constitutive disruption of the mouse PKN2 gene resulted in growth retardation and lethality before embryonic day (E) 10.5. PKN2-/- embryo did not undergo axial turning and showed insufficient closure of the neural tube. Mouse embryonic fibroblasts (MEFs) derived from PKN2-/- embryos at E9.5 failed to grow. Cre-mediated ablation of PKN2 in PKN2flox/flox MEFs obtained from E14.5 embryos showed impaired cell proliferation, and cell cycle analysis of these MEFs showed a decrease in S-phase population. Our results show that PKN2 is essential for mouse embryonic development and cell-autonomous proliferation of primary MEFs in culture. Comparison of the PKN2-/- phenotype with the phenotypes of PKN1 and PKN3 knockout strains suggests that PKN2 has distinct nonredundant functions in vivo, despite the structural similarity and evolutionary relationship among the three isoforms.
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Desenvolvimento Embrionário/fisiologia , Fibroblastos/citologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/genética , Feminino , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , GravidezRESUMO
Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high-density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break-up using botulinum hemagglutinin (HA), which specifically bound with E-cadherin and disrupted cell-cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E-cadherin-mediated cell-cell connections to facilitate the break-up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 106 cells ml-1 was obtained by aggregate break-up into small ones, which was three times higher than that with the conventional culture without aggregate break-up. Therefore, the temporary activity of HA for disrupting E-cadherin-mediated cell-cell connection was key to establishing a simple in situ method for hiPSC aggregate break-up in bioreactors, leading to high cell density in suspension culture.
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Comunicação Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Clostridium botulinum/metabolismo , Hemaglutininas/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Antígenos CD/metabolismo , Reatores Biológicos , Caderinas/metabolismo , Agregação Celular/efeitos dos fármacos , Contagem de Células , Meios de Cultura/análise , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , CinéticaRESUMO
Correction for 'Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1' by Keisuke Tanaka et al., Org. Biomol. Chem., 2018, 16, 4456-4463.