RESUMEN
The Zinc finger and SCAN domain containing 4 (ZSCAN4) protein, expressed transiently in pluripotent stem cells, gametes, and early embryos, extends telomeres, enhances genome stability, and improves karyotypes in mouse embryonic stem (mES) cells. To gain insights into the mechanism of ZSCAN4 function, we identified genome-wide binding sites of endogenous ZSCAN4 protein using ChIP-seq technology in mouse and human ES cells, where the expression of endogenous ZSCAN4 was induced by treating cells with retinoic acids or by overexpressing DUX4. We revealed that both mouse and human ZSCAN4 bind to the TGCACAC motif located in CA/TG microsatellite repeats, which are known to form unstable left-handed duplexes called Z-DNA that can induce double-strand DNA breaks and mutations. These ZSCAN4 binding sites are mostly located in intergenic and intronic regions of the genomes. By generating ZSCAN4 knockout in human ES cells, we showed that ZSCAN4 does not seem to be involved in transcriptional regulation. We also found that ectopic expression of mouse ZSCAN4 enhances the suppression of chromatin at ZSCAN4-binding sites. These results together suggest that some of the ZSCAN4 functions are mediated by binding to the error-prone regions in mouse and human genomes.
Asunto(s)
Genoma Humano , Factores de Transcripción , Humanos , Animales , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Dedos de Zinc , Repeticiones de Microsatélite , Proteínas de Unión al ADN/genéticaRESUMEN
Gaucher disease, the most prevalent metabolic storage disorder, is caused by mutations in the glucocerebrosidase gene GBA1, which lead to the accumulation of glucosylceramide (GlcCer) in affected cells. Gaucher disease type 1 (GD1), although defined as a nonneuronopathic subtype, is accompanied by an increased risk of Parkinson's disease. To gain insights into the association of progressive accumulation of GlcCer and the Parkinson's disease phenotypes, we generated dopaminergic (DA) neurons from induced pluripotent stem cells (iPSCs) derived from a GD1 patient and a healthy donor control, and measured GlcCer accumulation by liquid chromatography-mass spectrometry. We tested two DA neuron differentiation methods: a well-established method that mimics a step-wise developmental process from iPSCs to neural progenitor cells, and to DA neurons; and a synthetic mRNA-based method that overexpresses a transcription factor in iPSCs. GD1-specific accumulation of GlcCer was detected after 60 days of differentiation by the former method, whereas it was detected after only 10 days by the latter method. With this synthetic mRNA-based rapid differentiation method, we found that the metabolic defect in GD1 patient cells can be rescued by the overexpression of wild-type GBA1 or the treatment with an inhibitor for GlcCer synthesis. Furthermore, we detected the increased phosphorylation of α-synuclein, a biomarker for Parkinson's disease, in DA neurons derived from a GD1 patient, which was significantly decreased by the overexpression of wild-type GBA1. These results suggest that synthetic mRNA-based method accelerates the analyses of the pathological mechanisms of Parkinson's disease in GD1 patients and possibly facilitates drug discovery processes.
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Diferenciación Celular , Neuronas Dopaminérgicas , Enfermedad de Gaucher , Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , ARN Mensajero , Neuronas Dopaminérgicas/citología , Enfermedad de Gaucher/diagnóstico , Enfermedad de Gaucher/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Enfermedad de Parkinson/genética , Fenotipo , ARN Mensajero/genéticaRESUMEN
Transcription factors (TFs) play a pivotal role in determining cell states, yet our understanding of the causative relationship between TFs and cell states is limited. Here, we systematically examine the state changes of human pluripotent embryonic stem cells (hESCs) by the large-scale manipulation of single TFs. We establish 2,135 hESC lines, representing three clones each of 714 doxycycline (Dox)-inducible genes including 481 TFs, and obtain 26,998 microscopic cell images and 2,174 transcriptome datasets-RNA sequencing (RNA-seq) or microarrays-48 h after the presence or absence of Dox. Interestingly, the expression of essentially all the genes, including genes located in heterochromatin regions, are perturbed by these TFs. TFs are also characterized by their ability to induce differentiation of hESCs into specific cell lineages. These analyses help to provide a way of classifying TFs and identifying specific sets of TFs for directing hESC differentiation into desired cell types.
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Células Madre Embrionarias Humanas/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Células Madre Embrionarias Humanas/citología , Humanos , Análisis de la Célula Individual/métodosRESUMEN
The derivation of kidney tissues from human pluripotent stem cells (hPSCs) and its application for replacement therapy in end-stage renal disease have been widely discussed. Here we report that consecutive transfections of two sets of synthetic mRNAs encoding transcription factors can induce rapid and efficient differentiation of hPSCs into kidney tissues, termed induced nephron-like organoids (iNephLOs). The first set - FIGLA, PITX2, ASCL1 and TFAP2C, differentiated hPSCs into SIX2+SALL1+ nephron progenitor cells with 92% efficiency within 2 days. Subsequently, the second set - HNF1A, GATA3, GATA1 and EMX2, differentiated these cells into PAX8+LHX1+ pretubular aggregates in another 2 days. Further culture in both 2-dimensional and 3-dimensional conditions produced iNephLOs containing cells characterized as podocytes, proximal tubules, and distal tubules in an additional 10 days. Global gene expression profiles showed similarities between iNephLOs and the human adult kidney, suggesting possible uses of iNephLOs as in vitro models for kidneys.
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Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/citología , Riñón/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN Mensajero/genética , Factores de Transcripción/genética , Biomarcadores , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Linaje de la Célula/genética , Técnica del Anticuerpo Fluorescente , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunofenotipificación , Modelos Biológicos , Nefronas , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
BACKGROUND: Transplantation of pancreatic ß cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been developed for this purpose, lentivirus-mediated forced expression of transcription factors (TF)-PDX1 and NKX6.1-has been at the forefront for its relatively fast and straightforward approach. However, considering that such cells will be used for therapeutic purposes in the future, it is desirable to develop a procedure that does not leave any footprint on the genome, as any changes of DNAs could potentially be a source of unintended, concerning effects such as tumorigenicity. In this study, we attempted to establish a novel protocol for rapid and footprint-free hESC differentiation into a pancreatic endocrine lineage by using synthetic mRNAs (synRNAs) encoding PDX1 and NKX6.1. We also tested whether siPOU5F1, which reduces the expression of pluripotency gene POU5F1 (also known as OCT4), can enhance differentiation as reported previously for mesoderm and endoderm lineages. METHODS: synRNA-PDX1 and synRNA-NKX6.1 were synthesized in vitro and were transfected five times to hESCs with a lipofection reagent in a modified differentiation culture condition. siPOU5F1 was included only in the first transfection. Subsequently, cells were seeded onto a low attachment plate and aggregated by an orbital shaker. At day 13, the degree of differentiation was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry for endocrine hormones such as insulin, glucagon, and somatostatin. RESULTS: Both PDX1 and NKX6.1 expression were detected in cells co-transfected with synRNA-PDX1 and synRNA-NKX6.1 at day 3. Expression levels of insulin in the transfected cells at day 13 were 450 times and 14 times higher by qRT-PCR compared to the levels at day 0 and in cells cultured without synRNA transfection, respectively. Immunohistochemically, pancreatic endocrine hormones were not detected in cells cultured without synRNA transfection but were highly expressed in cells transfected with synRNA-PDX1, synRNA-NKX6.1, and siPOU5F1 at as early as day 13. CONCLUSIONS: In this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells.
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Proteínas de Homeodominio/genética , Células Madre Embrionarias Humanas/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/biosíntesis , ARN Mensajero/síntesis química , Transactivadores/genética , Diferenciación Celular , Ingeniería Celular/métodos , Células Cultivadas , Regulación de la Expresión Génica , Glucagón/biosíntesis , Proteínas de Homeodominio/metabolismo , Células Madre Embrionarias Humanas/citología , Humanos , Células Secretoras de Insulina/citología , Lípidos/química , Factor 3 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Somatostatina/biosíntesis , Transactivadores/metabolismo , TransfecciónRESUMEN
Direct generation of skeletal muscle cells from human pluripotent stem cells (hPSCs) would be beneficial for drug testing, drug discovery, and disease modelling in vitro. Here we show a rapid and robust method to induce myogenic differentiation of hPSCs by introducing mRNA encoding MYOD1 together with siRNA-mediated knockdown of POU5F1 (also known as OCT4 or OCT3/4). This integration-free approach generates functional skeletal myotubes with sarcomere-like structure and a fusion capacity in several days. The POU5F1 silencing facilitates MYOD1 recruitment to the target promoters, which results in the significant activation of myogenic genes in hPSCs. Furthermore, deep sequencing transcriptome analyses demonstrated that POU5F1-knockdown upregulates the genes associated with IGF- and FGF-signaling and extracellular matrix that may also support myogenic differentiation. This rapid and direct differentiation method may have potential applications in regenerative medicine and disease therapeutics for muscle disorders such as muscular dystrophy.
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Diferenciación Celular/genética , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Proteína MioD/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Modelos Biológicos , Desarrollo de Músculos/genéticaRESUMEN
Dry eye disease is the most prevalent pathological condition in aging eyes. One potential therapeutic strategy is the transplantation of lacrimal glands, generated in vitro from pluripotent stem cells such as human embryonic stem cells, into patients. One of the preceding requirements is a method to differentiate human embryonic stem cells into lacrimal gland epithelium cells. As the first step for this approach, this study aims to identify a set of transcription factors whose overexpression can promote the differentiation of human embryonic stem cells into lacrimal gland epithelium-like cells. We performed microarray analyses of lacrimal glands and lacrimal glands-related organs obtained from mouse embryos and adults, and identified transcription factors enriched in lacrimal gland epithelium cells. We then transfected synthetic messenger RNAs encoding human orthologues of these transcription factors into human embryonic stem cells and examined whether the human embryonic stem cells differentiate into lacrimal gland epithelium-like cells by assessing cell morphology and marker gene expression. The microarray analysis of lacrimal glands tissues identified 16 transcription factors that were enriched in lacrimal gland epithelium cells. We focused on three of the transcription factors, because they are expressed in other glands such as salivary glands and are also known to be involved in the development of lacrimal glands. We tested the overexpression of various combinations of the three transcription factors and PAX6, which is an indispensable gene for lacrimal glands development, in human embryonic stem cells. Combining PAX6, SIX1, and FOXC1 caused significant changes in morphology, i.e., elongated cell shape and increased expression (both RNAs and proteins) of epithelial markers such as cytokeratin15, branching morphogenesis markers such as BARX2, and lacrimal glands markers such as aquaporin5 and lactoferrin. We identified a set of transcription factors enriched in lacrimal gland epithelium cells and demonstrated that the simultaneous overexpression of these transcription factors can differentiate human embryonic stem cells into lacrimal gland epithelium-like cells. This study suggests the possibility of lacrimal glands regeneration from human pluripotent stem cells.
RESUMEN
Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons. To this end, we established hESC lines in which each of these TFs can be overexpressed by the doxycycline-inducible piggyBac vector. The overexpression of any of these five TFs indeed caused a rapid and rather uniform differentiation of hESCs, which were identified as neurons based on their morphologies, qRT-PCR, and immunohistochemistry. Furthermore, calcium-imaging analyses and patch clamp recordings demonstrated that these differentiated cells are electrophysiologically functional. Interestingly, neural differentiations occurred despite the cell culture conditions that rather promote the maintenance of the undifferentiated state. These results indicate that over-expression of each of these five TFs can override the pluripotency-specific gene network and force hESCs to differentiate into neurons.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias Humanas/citología , Neuronas/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transgenes/genética , Células Cultivadas , Células Madre Embrionarias Humanas/metabolismo , Humanos , Neuronas/metabolismoRESUMEN
Human pluripotent stem cells (hPSCs) have the capacity to differentiate into essentially all cell types in the body. Such differentiation can be directed to specific cell types by appropriate cell culture conditions or overexpressing lineage-defining transcription factors (TFs). Especially, for the activation of myogenic program, early studies have shown the effectiveness of enforced expression of TFs associated with myogenic differentiation, such as PAX7 and MYOD1. However, the efficiency of direct differentiation was rather low, most likely due to chromatin features unique to hPSCs, which hinder the access of TFs to genes involved in muscle differentiation. Indeed, recent studies have demonstrated that ectopic expression of epigenetic-modifying factors such as a histone demethylase and an ATP-dependent remodeling factor significantly enhances myogenic differentiation from hPSCs. In this article, we review the recent progress for in vitro generation of skeletal muscles from hPSCs through forced epigenetic and transcriptional manipulation.
RESUMEN
MOTIVATION: The analysis of RNA-Seq data from individual differentiating cells enables us to reconstruct the differentiation process and the degree of differentiation (in pseudo-time) of each cell. Such analyses can reveal detailed expression dynamics and functional relationships for differentiation. To further elucidate differentiation processes, more insight into gene regulatory networks is required. The pseudo-time can be regarded as time information and, therefore, single-cell RNA-Seq data are time-course data with high time resolution. Although time-course data are useful for inferring networks, conventional inference algorithms for such data suffer from high time complexity when the number of samples and genes is large. Therefore, a novel algorithm is necessary to infer networks from single-cell RNA-Seq during differentiation. RESULTS: In this study, we developed the novel and efficient algorithm SCODE to infer regulatory networks, based on ordinary differential equations. We applied SCODE to three single-cell RNA-Seq datasets and confirmed that SCODE can reconstruct observed expression dynamics. We evaluated SCODE by comparing its inferred networks with use of a DNaseI-footprint based network. The performance of SCODE was best for two of the datasets and nearly best for the remaining dataset. We also compared the runtimes and showed that the runtimes for SCODE are significantly shorter than for alternatives. Thus, our algorithm provides a promising approach for further single-cell differentiation analyses. AVAILABILITY AND IMPLEMENTATION: The R source code of SCODE is available at https://github.com/hmatsu1226/SCODE. CONTACT: hirotaka.matsumoto@riken.jp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Diferenciación Celular/genética , Redes Reguladoras de Genes , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Algoritmos , Animales , Humanos , Ratones , Análisis de la Célula Individual/métodosRESUMEN
The mechanisms of immunoactivation by salt are now becoming clearer. However, those of immunosuppression remain unknown. Since clinical evidence indicates that salt protects proximal tubules from injury, we investigated mechanisms responsible for salt causing immunosuppression in proximal tubules. We focused on cytokine-related gene expression profiles in kidneys of mice fed a high salt diet using microarray analysis and found that both an interferon gamma (IFNγ) inducible chemokine, chemokine (C-X-C motif) ligand 9 (CXCL9), and receptor, CXCR3, were suppressed. We further revealed that a high salt concentration suppressed IFNγ inducible chemokines in HK2 proximal tubular cells. Finally, we demonstrated that a high salt concentration decreased IFNGR1 expression in the basolateral membrane of HK2 cells, leading to decreased phosphorylation of activation sites of Janus kinase 1 (JAK1) and Signal Transducers and Activator of Transcription 1 (STAT1), activators of chemokines. JAK inhibitor canceled the effect of a high salt concentration on STAT1 and chemokines, indicating that the JAK1-STAT1 signaling pathway is essential for this mechanism. In conclusion, a high salt concentration suppresses IFNγ-JAK1-STAT1 signaling pathways and chemokine expressions in proximal tubules. This finding may explain how salt ameliorates proximal tubular injury and offer a new insight into the linkage between salt and immunity.
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Quimiocinas/genética , Células Epiteliales/efectos de los fármacos , Interferón gamma/farmacología , Transducción de Señal/efectos de los fármacos , Cloruro de Sodio/farmacología , Animales , Antivirales/farmacología , Línea Celular , Quimiocinas/metabolismo , Células Epiteliales/metabolismo , Humanos , Interferón gamma/metabolismo , Janus Quinasa 1/metabolismo , Túbulos Renales Proximales/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Receptor de Interferón gammaRESUMEN
Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs.
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Diferenciación Celular/genética , Neuronas Motoras/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/genética , Biomarcadores/metabolismo , Forma de la Célula , Humanos , Canales Iónicos/metabolismo , Cinética , Neuronas Motoras/metabolismo , Neurogénesis/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Mouse Zinc finger and SCAN domain containing 4 (Zscan4) is encoded in multiple copies of Zscan4 genes, which are expressed in late two-cell stage preimplantation embryos and in 1-5% of the embryonic stem (ES) cell population at a given time. Due to the highly identical nucleotide sequences of multiple copies of Zscan4 paralogs and pseudogenes in the mouse Zscan4 genomic cluster, previous analyses have been done using exogenous transgenes under the regulation of Zscan4c promoter. In this manuscript, we generated knock-in mouse ES cell lines and mouse lines, in which the expression of endogenous Zscan4c, one of the Zscan4 genes, can be specifically monitored with a green fluorescent protein variant, Emerald. Interestingly, we found that only â¼30% of Zscan4-immunopositive ES cells were Emerald positive, suggesting that even when the Zscan4 locus is active, not all Zscan4 genes are expressed synchronously. We also carried out mass spectrometry of protein complexes associated with endogenous Zscan4 proteins. Taken together, our genetic engineering at an endogenous Zscan4c gene provides the first clue for the expression and function of each gene copy of Zscan4 locus in a physiological context.
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Blastocisto/metabolismo , Proteínas Cromosómicas no Histona/genética , Regulación del Desarrollo de la Expresión Génica , Sitios Genéticos , Células Madre Embrionarias de Ratones/metabolismo , Sistemas de Lectura Abierta/genética , Factores de Transcripción/genética , Animales , Blastocisto/efectos de los fármacos , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Sustitución del Gen , Marcación de Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Tretinoina/farmacologíaRESUMEN
Mouse zinc finger and SCAN domain containing 4 (Zscan4) proteins, which are encoded by multiple copies of Zscan4 genes, are expressed specifically in preimplantation embryos in vivo and embryonic stem (ES) cells in vitro. However, the expression patterns of mouse Zscan4 in vivo have been largely elusive. Here, we show that Zscan4 proteins are expressed in adult ovaries and testes. In ovaries, Zscan4 proteins were detected in germinal vesicle (GV) stage oocytes in antral follicles, indicating that Zscan4 genes are activated during the diplotene/dictyate stage in meiotic prophase I. Remarkably, Zscan4 showed different spatial localization patterns between two distinct GV oocytes, which can be distinguished by global chromatin organization-surrounded nucleolus (SN) and non-surrounded nucleolus (NSN). These spatiotemporal differences in Zscan4 localizations correlated with the transition of RNA polymerase II-mediated transcriptional status during GV oocyte maturation. In testes, Zscan4 proteins were detected in spermatocytes at late pachytene/diplotene stages and in Sertoli cells. These results suggest that Zscan4 may play critical roles during late meiotic prophase in both males and females.
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Proteínas Cromosómicas no Histona/metabolismo , Profase Meiótica I , Oogénesis , Espermatogénesis , Factores de Transcripción/metabolismo , Animales , Blastocisto/citología , Blastocisto/metabolismo , Proteínas Cromosómicas no Histona/genética , Femenino , Masculino , Profase Meiótica I/genética , Ratones Endogámicos C57BL , Oogénesis/genética , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células de Sertoli/citología , Células de Sertoli/metabolismo , Espermatogénesis/genética , Espermatozoides/citología , Espermatozoides/metabolismo , Testículo/citología , Testículo/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
KEY POINTS: The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3- concentrations as high as 140 mm during hormonal stimulation. A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea-pig pancreatic ducts. The model was readily able to secrete 140 mm HCO3- . Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers. We conclude that the main requirement for secreting high HCO3- concentrations is to minimize the secretion of Cl- ions. These findings help to clarify the mechanism responsible for pancreatic HCO3- secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease. ABSTRACT: A computational model of guinea-pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3- ions are secreted at concentrations in excess of 140 mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least-squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP-stimulated secretion were well replicated by increasing the activities of the basolateral Na+ -HCO3- cotransporter (NBC1) and apical Cl- /HCO3- exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl- and HCO3- permeabilities (CFTR), and reducing the activity of the basolateral Cl- /HCO3- exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted â¼140 mm HCO3- at a rate of â¼3 nl min-1 mm-2 , which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl- /HCO3- exchange via SLC26A6 at the apical membrane were able to support a HCO3- -rich secretion. Raising the HCO3- /Cl- permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3- concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by â¼80% was essential in minimizing the intracellular Cl- concentration following cAMP stimulation and thereby maximizing the secreted HCO3- concentration. The addition of a basolateral Na+ -K+ -2Cl- cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl- and resulted in a lower secreted HCO3- concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl- secretion is the main requirement for secreting 140 mm HCO3- .
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Bicarbonatos/metabolismo , Cloruros/metabolismo , Conductos Pancreáticos/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Membrana Celular/fisiología , Epitelio/metabolismo , Cobayas , Potenciales de la Membrana , Proteínas de Transporte de Membrana/metabolismo , Modelos BiológicosRESUMEN
Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings.
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Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Apoptosis/genética , Apoptosis/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Inmunoprecipitación de Cromatina , Expresión Génica Ectópica/genética , Expresión Génica Ectópica/fisiología , Epigénesis Genética/genética , Hepatocitos/metabolismo , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Immunoblotting , Histona Demetilasas con Dominio de Jumonji/genética , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/metabolismo , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-κB ligand (RANKL), antagonizes RANKL's osteoclastogenic function in bone. We previously demonstrated that systemic administration of lipopolysaccharide (LPS) to mice elevates OPG levels and reduces RANKL levels in peripheral blood. Here, we show that mice infected with Salmonella, Staphylococcus, Mycobacteria or influenza virus also show elevated serum OPG levels. We then asked whether OPG upregulation following microbial invasion had an effect outside of bone. To do so, we treated mice with LPS and observed OPG production in pancreas, especially in ß-cells of pancreatic islets. Insulin release following LPS administration was enhanced in mice lacking OPG, suggesting that OPG inhibits insulin secretion under acute inflammatory conditions. Consistently, treatment of MIN6 pancreatic ß-cells with OPG decreased their insulin secretion following glucose stimulation in the presence of LPS. Finally, our findings suggest that LPS-induced OPG upregulation is mediated in part by activator protein (AP)-1 family transcription factors, particularly Fos proteins. Overall, we report that acute microbial infection elevates serum OPG, which maintains ß-cell homeostasis by restricting glucose-stimulated insulin secretion, possibly preventing microbe-induced exhaustion of ß-cell secretory capacity.
Asunto(s)
Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Osteoprotegerina/metabolismo , Animales , Línea Celular , Femenino , Homeostasis/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones , Ratones Mutantes , Mycobacterium/fisiología , Proteínas Oncogénicas v-fos/metabolismo , Osteoprotegerina/farmacología , Ligando RANK/metabolismo , Salmonella/fisiología , Staphylococcus/fisiología , Factor de Transcripción AP-1/metabolismoRESUMEN
Mouse embryonic stem cells (mESCs) have a remarkable capacity to maintain normal genome stability and karyotype in culture. We previously showed that infrequent bursts of Zscan4 expression (Z4 events) are important for the maintenance of telomere length and genome stability in mESCs. However, the molecular details of Z4 events remain unclear. Here we show that Z4 events involve unexpected transcriptional derepression in heterochromatin regions that usually remain silent. During a Z4 event, we see rapid derepression and rerepression of heterochromatin leading to a burst of transcription that coincides with transient histone hyperacetylation and DNA demethylation, clustering of pericentromeric heterochromatin around the nucleolus, and accumulation of activating and repressive chromatin remodelling complexes. This heterochromatin-based transcriptional activity suggests that mESCs may maintain their extraordinary genome stability at least in part by transiently resetting their heterochromatin.
Asunto(s)
Epigénesis Genética , Heterocromatina/genética , Células Madre Embrionarias de Ratones/metabolismo , Homeostasis del Telómero/genética , Factores de Transcripción/genética , Acetilación , Animales , Nucléolo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Metilación de ADN , Inestabilidad Genómica , Histonas/metabolismo , Ratones , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
Although cystic fibrosis is rare in Japanese, measurement of sweat Cl(-) has suggested mild dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) in some patients with chronic pancreatitis. In the present study, we have investigated the association of CFTR variants and chronic pancreatitis in Japanese and the functional characteristics of a Japanese- and pancreatitis-specific CFTR variant, L1156F. Seventy patients with alcoholic chronic pancreatitis, 18 patients with idiopathic chronic pancreatitis, and 180 normal subjects participated. All exons and their boundaries and promoter region of the CFTR gene were sequenced. Human embryonic kidney-293 cells were transfected with three CFTR variants (M470V, L1156F, and M470V+L1156F), and the protein expression was examined. Xenopus laevis oocytes were injected with the CFTR variants, and bicarbonate (HCO3 (-)) transport activity was examined. CFPAC-1 cells were transfected with the CFTR variants and Cl(-)/HCO3 (-) exchange activity was examined. Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Cystic fibrosis-causing mutations were not found. The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P < 0.01) higher than those in normal subjects (0.6 and 1.9%). L1156F was linked with a worldwide CFTR variant, M470V. Combination of M470V and L1156F significantly reduced CFTR expression to â¼60%, impaired CFTR-mediated HCO3 (-)/Cl(-) transport activity to 50-60%, and impaired CFTR-coupled Cl(-)/HCO3 (-) exchange activity to 20-30%. The data suggest that the Japanese-specific CFTR variant L1156F causes mild dysfunction of CFTR and increases the risk of alcoholic chronic pancreatitis in Japanese.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Mutación Missense , Pancreatitis Alcohólica/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Bicarbonatos/metabolismo , Estudios de Casos y Controles , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Exones , Femenino , Frecuencia de los Genes , Células HEK293 , Humanos , Transporte Iónico , Japón , Masculino , Persona de Mediana Edad , XenopusRESUMEN
Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols.