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1.
Cell ; 160(1-2): 253-68, 2015 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-25543152

RESUMEN

Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.


Asunto(s)
Diferenciación Celular , Células Germinativas/citología , Factores de Transcripción SOXF/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Línea Celular Tumoral , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Células Germinativas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Represoras/metabolismo , Seminoma/metabolismo , Análisis de Secuencia de ARN
2.
Cell ; 142(5): 787-99, 2010 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-20813264

RESUMEN

The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.


Asunto(s)
Blastocisto , Quimera/embriología , Páncreas/citología , Páncreas/embriología , Células Madre Pluripotentes , Animales , Diabetes Mellitus/inducido químicamente , Diabetes Mellitus/terapia , Desarrollo Embrionario , Técnicas de Sustitución del Gen , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos , Organogénesis , Ratas , Ratas Wistar , Transactivadores/genética
3.
Development ; 148(23)2021 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-34874452

RESUMEN

Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.


Asunto(s)
Diferenciación Celular , Embrión de Mamíferos/metabolismo , Estratos Germinativos/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Bovinos , Embrión de Mamíferos/citología , Estratos Germinativos/citología , Ganado , Células Madre Pluripotentes/citología , Ovinos , Especificidad de la Especie , Porcinos
4.
Int J Clin Oncol ; 29(5): 602-611, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38418804

RESUMEN

BACKGROUND: Enfortumab vedotin is a novel antibody-drug conjugate used as a third-line therapy for the treatment of urothelial cancer. We aimed to elucidate the effect of enfortumab vedotin-related peripheral neuropathy on its efficacy and whether enfortumab vedotin-induced early electrophysiological changes could be associated with peripheral neuropathy onset. METHODS: Our prospective multicenter cohort study enrolled 34 patients with prior platinum-containing chemotherapy and programmed cell death protein 1/ligand 1 inhibitor-resistant advanced urothelial carcinoma and received enfortumab vedotin. The best overall response, progression-free survival, overall survival, and safety were assessed. Nerve conduction studies were also performed in 11 patients. RESULTS: The confirmed overall response rate and disease control rate were 52.9% and 73.5%, respectively. The median overall progression-free survival and overall survival were 6.9 and 13.5 months, respectively, during a median follow-up of 8.6 months. The patients with disease control had significantly longer treatment continuation and overall survival than did those with uncontrolled disease. Peripheral neuropathy occurred in 12.5% of the patients. The overall response and disease control rates were 83.3% and 100%, respectively: higher than those in patients without peripheral neuropathy (p = 0.028 and p = 0.029, respectively). Nerve conduction studies indicated that enfortumab vedotin reduced nerve conduction velocity more markedly in sensory nerves than in motor nerves and the lower limbs than in the upper limbs, with the sural nerve being the most affected in the patients who developed peripheral neuropathy (p = 0.011). CONCLUSION: Our results indicated the importance of focusing on enfortumab vedotin-induced neuropathy of the sural nerve to maximize efficacy and improve safety.


Asunto(s)
Anticuerpos Monoclonales , Enfermedades del Sistema Nervioso Periférico , Humanos , Masculino , Femenino , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Anciano , Estudios Prospectivos , Persona de Mediana Edad , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/efectos adversos , Anciano de 80 o más Años , Conducción Nerviosa/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patología , Carcinoma de Células Transicionales/tratamiento farmacológico , Supervivencia sin Progresión , Neoplasias Urológicas/tratamiento farmacológico , Neoplasias Urológicas/patología
5.
Development ; 147(4)2020 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-32001439

RESUMEN

Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying Prdm14, a unique marker and an essential PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until the sex determination stage in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that the crucial role of Prdm14 in PGC specification is conserved between rat and mice, by analyzing Prdm14-deficient rat embryos. Notably, loss of Prdm14 completely abrogates the PGC program, as demonstrated by failure of the maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the post-implantation epiblast and all PGC stages in rat to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional timeline for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved versus species-specific features for germline development in mammals.


Asunto(s)
Proteínas de Unión al ADN/genética , Células Germinativas/citología , Proteínas de Unión al ARN/genética , Factores de Transcripción/genética , Animales , Cruzamientos Genéticos , Proteínas de Unión al ADN/fisiología , Femenino , Gástrula/fisiología , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Heterocigoto , Masculino , Ratones , Proteínas de Unión al ARN/fisiología , Ratas , Procesos de Determinación del Sexo , Factores de Transcripción/fisiología , Transcripción Genética
6.
Nature ; 546(7658): 416-420, 2017 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-28607482

RESUMEN

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during weeks 2-3 of early post-implantation development. Using in vitro models of hPGC induction, recent studies have suggested that there are marked mechanistic differences in the specification of human and mouse PGCs. This may be due in part to the divergence in their pluripotency networks and early post-implantation development. As early human embryos are not accessible for direct study, we considered alternatives including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs originate from the posterior pre-primitive-streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. We use this model together with human and monkey in vitro models simulating peri-gastrulation development to show the conserved principles of epiblast development for competency for primordial germ cell fate. This process is followed by initiation of the epigenetic program and regulated by a balanced SOX17-BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models provides synthetic insights into early human development.


Asunto(s)
Diferenciación Celular , Desarrollo Embrionario , Células Germinativas/citología , Macaca fascicularis/embriología , Modelos Biológicos , Células Madre Pluripotentes/citología , Porcinos/embriología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Linaje de la Célula , Cuerpos Embrioides/citología , Epigénesis Genética , Femenino , Gastrulación , Dosificación de Gen , Células Germinativas/metabolismo , Estratos Germinativos/citología , Humanos , Técnicas In Vitro , Masculino , Modelos Animales , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Línea Primitiva/citología , Proteínas Represoras/genética , Factores de Transcripción SOXF/genética , Vía de Señalización Wnt
7.
Nature ; 542(7640): 191-196, 2017 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-28117444

RESUMEN

Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.


Asunto(s)
Diabetes Mellitus Experimental/terapia , Xenoinjertos/fisiología , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/fisiología , Organogénesis , Animales , Blastocisto/citología , Blastocisto/metabolismo , Glucemia/metabolismo , Quimera , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Femenino , Xenoinjertos/inmunología , Proteínas de Homeodominio , Islotes Pancreáticos/citología , Islotes Pancreáticos/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Masculino , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/trasplante , Ratas , Factores de Tiempo , Transactivadores/deficiencia
8.
Pediatr Int ; 65(1): e15700, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37991185

RESUMEN

BACKGROUND: In Japan, the vaccination rate against the SARS-CoV-2 vaccine for children was low. Therefore, in this study we investigated the factors influencing guardians' decision-making regarding vaccination of their children. METHODS: From November 1, 2022 to March 31, 2023, pediatric clinics, departments, and midwifery clinics in Saitama Prefecture requested guardians of children under the age of 15 to complete an online questionnaire. RESULTS: Responses were obtained from 894 guardians of children aged 6 months to 15 years; 142 had had one of their children vaccinated at least once and 629 had not had any of their children vaccinated. Among guardians who had not had any of their children vaccinated, "the Age of children" was significantly younger (p < 0.001) and "Prevalence" (p < 0.001), "Free vaccination" (p < 0.001), and "Intentions of national and local governments" (p = 0.005) were selected as reasons significantly less frequently in comparison to guardians who had vaccinated their children. "Japanese adverse reactions" (p < 0.001), "Japanese effectiveness" (p < 0.001), "Adverse reactions" (p < 0.001), "History of adverse reactions" (p < 0.001), and "Reputation of friends" (p = 0.006) were selected significantly more frequently by guardians who had not had any of their children vaccinated. CONCLUSIONS: Guardians who had had one of their children vaccinated at least once emphasized the importance of prevalence and free vaccination. On the other hand, guardians who had not had any of their children vaccinated placed particular importance on adverse reactions and the Japanese data on effectiveness. To guide the decision-making of guardians, it is necessary to quickly collect and publish data on adverse reactions and effectiveness, particularly in Japanese individuals, so that citizens can decide whether to vaccinate themselves and their children.


Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Vacunación , Niño , Humanos , COVID-19/epidemiología , COVID-19/prevención & control , Vacunas contra la COVID-19/administración & dosificación , Japón , SARS-CoV-2 , Vacunación/psicología , Toma de Decisiones
9.
Curr Issues Mol Biol ; 44(11): 5474-5484, 2022 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-36354682

RESUMEN

Glucagon-like peptide-1 receptor agonist (GLP-1RA) has been clinically proven to protect endothelial function. Previously, we demonstrated that endothelial NO synthase (eNOS) was activated by high-density lipoprotein (HDL) via its scavenger receptor of the B class/human homologue of SR-BI, CD36 and LIMPII analogous-1(hSR-BI/CLA-1). Here, we investigated the effect of GLP-1RA and exendin-4 on the expression of hSR-BI/CLA-1 in HUVECs. Our results confirmed that GLP-1R was expressed in HUVECs by PCR and exendin-4 significantly enhanced HDL-induced eNOS activation. Next, exendin-4 increased the expression of hSR-BI/CLA-1 and a blockade of GLP-1R cancelled this effect. Further, the hSR-BI/CLA-1 transcriptional activity was enhanced by exendin-4, which was diminished by the inhibition of AMPK or dominant-negative AMPK-α-subunit. Moreover, AMPK was phosphorylated by the activation of GLP-1R. Next, ChIP assay demonstrated that exendin-4 increased the FoxO1-binding in the hSR-BI/CLA-1 promoter by upregulation of FoxO1. Mutation of FoxO1-binding or silencing of FoxO1 cancelled the effect of exendin-4 on hSR-BI/CLA-1 expression. Exendin-4 reduced FoxO1 phosphorylation and induced its nuclear accumulation, while this effect was altered by the blocking of GLP-1R or inhibition of AMPK pathway. In summary, our results proved that exendin-4 increased hSR-BI/CLA-1 expression via the AMPK/FoxO1 pathway to activate eNOS, providing a basic mechanism underlining the protective effect of GLP-1RA on endothelial function.

10.
Mol Reprod Dev ; 89(3): 129-132, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35170139

RESUMEN

Gene editing in mammalian zygotes enables us to generate genetically modified animals rapidly and efficiently. In this study, we compare multiple gene targeting strategies in rat zygotes by generating a novel knock-in reporter rat line to visualize the expression pattern of transcription factor AP-2 gamma (Tfap2c). The targeting vector is designed to replace the stop codon of Tfap2c with T2A-tdTomato sequence. We show that the combination of electroporation-mediated transduction of CRISPR/Cas9 components with adeno-associated virus-mediated transduction of the targeting vector is the most efficient in generating the targeted rat line. The Tfap2c-T2A-tdTomato fluorescence reflects the endogenous expression pattern of Tfap2c in preimplantation embryo, germline, placenta, and forebrain during rat embryo development. The reporter line generated here will be a reliable resource for identifying and purifying Tfap2c expressing cells in rats, and the gene targeting strategy we used can be widely applied for generating desired animals.


Asunto(s)
Sistemas CRISPR-Cas , Dependovirus , Animales , Dependovirus/genética , Femenino , Edición Génica , Técnicas de Sustitución del Gen , Marcación de Gen , Proteínas Luminiscentes , Mamíferos/genética , Embarazo , Ratas , Cigoto/metabolismo , Proteína Fluorescente Roja
11.
Catheter Cardiovasc Interv ; 100(5): 868-876, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36073020

RESUMEN

OBJECTIVES: To assess the inter methodological agreement of membrane septum (MS) length measurement and additive value for risk stratification of new pacemaker implantation (PMI) over the established predictors after transcatheter aortic valve replacement (TAVR). BACKGROUND: Recent studies have suggested MS length and implantation depth (ID) as predictors for PMI after TAVR. However, the measurement of MS length is neither uniform nor validated in different cohort. METHODS: We retrospectively analyzed patients who underwent TAVR at five centers. The MS length was measured by two previously proposed methods (coronal and annular view method). Predictive ability of risk factors, including MS length and ID, for new PMI within 30 days after TAVR were evaluated. RESULTS: Among 754 patients of study population, 31 patients (4.1%) required new PMI within 30 days of TAVR. There was a weak correlation (ρ = 0.47) and a poor agreement between the two methods. The ID and the difference between MS length and ID (ΔMSID), were independent predictors for new PMI, whereas MS length alone was not. Further, for predicting new PMI after TAVR, discrimination performance was not significantly improved when MS length was added to the model with ID alone (integrated discrimination improvement = 0, p= 0.99; continuous net-reclassification improvement = 0.10, p= 0.62). CONCLUSIONS: External validity and predictive accuracy of MS length for PMI after TAVR were not sufficient to provide better risk stratification over the established predictors in our cohort. Moreover, the ID and ΔMSID, but not MS length alone, are predictive of future PMI after TAVR.


Asunto(s)
Estenosis de la Válvula Aórtica , Prótesis Valvulares Cardíacas , Marcapaso Artificial , Reemplazo de la Válvula Aórtica Transcatéter , Humanos , Reemplazo de la Válvula Aórtica Transcatéter/efectos adversos , Estenosis de la Válvula Aórtica/diagnóstico por imagen , Estenosis de la Válvula Aórtica/cirugía , Estenosis de la Válvula Aórtica/etiología , Válvula Aórtica/diagnóstico por imagen , Válvula Aórtica/cirugía , Estudios Retrospectivos , Reproducibilidad de los Resultados , Estimulación Cardíaca Artificial/efectos adversos , Resultado del Tratamiento , Factores de Riesgo
12.
Nat Rev Genet ; 17(10): 585-600, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27573372

RESUMEN

Primordial germ cells (PGCs), the precursors of sperm and eggs, are established in perigastrulation-stage embryos in mammals. Signals from extra-embryonic tissues induce a unique gene regulatory network in germline-competent cells for PGC specification. This network also initiates comprehensive epigenome resetting, including global DNA demethylation and chromatin reorganization. Mouse germline development has been studied extensively, but the extent to which such knowledge applies to humans was unclear. Here, we review the latest advances in human PGC specification and epigenetic reprogramming. The overall developmental dynamics of human and mouse germline cells appear to be similar, but there are crucial mechanistic differences in PGC specification, reflecting divergence in the regulation of pluripotency and early development.


Asunto(s)
Metilación de ADN , Epigénesis Genética/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Células Germinativas , Animales , Humanos , Ratones , Transducción de Señal
13.
Nature ; 529(7586): 403-407, 2016 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-26751055

RESUMEN

Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Células Germinativas/citología , Células Germinativas/metabolismo , Estratos Germinativos/citología , Proteínas de Homeodominio/metabolismo , Células Madre Embrionarias de Ratones/citología , Factores de Transcripción/genética , Activinas/farmacología , Animales , Proteína Morfogenética Ósea 4/metabolismo , Diferenciación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN , Epigénesis Genética , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación del Desarrollo de la Expresión Génica , Genoma/genética , Estratos Germinativos/efectos de los fármacos , Estratos Germinativos/metabolismo , Proteínas de Homeodominio/antagonistas & inhibidores , Masculino , Ratones , Células Madre Embrionarias de Ratones/efectos de los fármacos , Proteína Homeótica Nanog , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Unión Proteica , Proteínas de Unión al ARN , Factores de Transcripción SOXB1/metabolismo , Factor de Transcripción AP-2/genética , Factor de Transcripción AP-2/metabolismo , Factores de Transcripción/metabolismo
14.
Development ; 145(16)2018 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-30037844

RESUMEN

In mice, primordial germ cells (PGCs), the precursors of eggs and sperm, originate from pregastrulation postimplantation embryos. By contrast, the origin of human PGCs (hPGCs) has been less clear and has been difficult to study because of the technical and ethical constraints that limit direct studies on human embryos. In recent years, however, in vitro simulation models using human pluripotent stem cells, together with surrogate non-rodent mammalian embryos, have provided insights and experimental approaches to address this issue. Here, we review these studies, which suggest that the posterior epiblast and/or the nascent amnion in pregastrulation human embryos is a likely source of hPGCs, and that a different gene regulatory network controls PGCs in humans compared with in the mouse. Such studies on the origins and mechanisms of hPGC specification prompt further consideration of the somatic cell fate decisions that occur during early human development.


Asunto(s)
Desarrollo Embrionario/fisiología , Células Germinativas/fisiología , Células Madre Embrionarias Humanas/fisiología , Animales , Diferenciación Celular/genética , Embrión de Mamíferos , Células Madre Embrionarias/fisiología , Epigénesis Genética , Redes Reguladoras de Genes , Estratos Germinativos/embriología , Estratos Germinativos/fisiología , Células Madre Embrionarias Humanas/citología , Humanos , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología
15.
Development ; 145(20)2018 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-30185410

RESUMEN

The precise control of gene expression by transcription factor networks is crucial to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here, we modify our targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify genome-wide protein/DNA interactions in 100 to 1000 times fewer cells than ChIP-based approaches. We mapped the binding sites of two key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system for elucidating primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its crucial role in PGCLC development. We show that MaTaDa is a sensitive and accurate method for assessing cell type-specific transcription factor binding in limited numbers of cells.


Asunto(s)
Metilación de ADN/genética , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Cromatina/metabolismo , Proteínas de Unión al ADN , Genoma , Células Germinativas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Unión Proteica , Proteínas de Unión al ARN
16.
Mol Hum Reprod ; 26(6): 441-451, 2020 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-32333783

RESUMEN

Concentrations of 2-methoxyestradiol (2ME2), a principal metabolite of estradiol, are significantly lower in women with severe preeclampsia. Nitric oxide (NO) released by endothelial nitric oxide synthase (eNOS) plays an important role in regulating cardiovascular homeostasis. Importantly, high-density lipoprotein (HDL) stimulates eNOS activity via endothelial human scavenger receptor class B type I (hSR-BI/CLA-1). Here, we aimed to determine the effect of 2ME2 on hSR-BI/CLA-1 expression in human umbilical vein endothelial cells (HUVECs). hSR-BI/CLA-1 expression was measured by real-time PCR, western blotting and reporter gene assays; eNOS activity was assessed by the measurement of eNOS phosphorylation. Both the mRNA and protein concentrations of hSR-BI/CLA-1 were significantly increased by 2ME2 in HUVECs. 2ME2 also dose-dependently increased the transcriptional activity of the hSR-BI/CLA-1 promoter. The effect of 2ME2 treatment on the promoter activity of hSR-BI/CLA-1 was abrogated by treatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase, as was the increase in HDL-induced eNOS activation. Notably, constitutively active Akt increased the activity of the hSR-BI/CLA-1 promoter, whereas dominant-negative Akt abolished the effect of 2ME2 treatment on hSR-BI/CLA-1 promoter activity. The nuclear Sp1 protein concentration was significantly increased by exposure to 2ME2 and Sp1 overexpression increased the promoter activity of the hSR-BI/CLA gene. Furthermore, knockdown of Sp1 inhibited the effect of 2ME2 treatment on hSR-BI/CLA-1 protein expression. These results indicate that 2ME2 treatment increases HDL-dependent eNOS phosphorylation by upregulating endothelial hSR-BI/CLA-1 expression, suggesting that 2ME2 has a potential therapeutic value in the treatment of preeclampsia.


Asunto(s)
2-Metoxiestradiol/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , 2-Metoxiestradiol/uso terapéutico , Western Blotting , Inmunoprecipitación de Cromatina , Femenino , Humanos , Lipoproteínas HDL/metabolismo , Óxido Nítrico/metabolismo , Preeclampsia/tratamiento farmacológico , Preeclampsia/metabolismo , Embarazo
17.
Gastric Cancer ; 23(4): 760-764, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32146645

RESUMEN

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract; most of them have gain-of-function mutations of the KIT gene. There have been rare cases of families with multiple GISTs, that had autosomal dominant germline KIT mutations. Here, we present a case of multiple GISTs caused by a novel germline KIT mutation. Intraoperatively, the main tumor was present in the body of the stomach, and multiple small nodules were detected mainly in the upper and middle part of the gastric wall; several nodules were also present in the small bowel wall. The main tumor and surrounding nodules were resected. DNA sequencing of the tumor tissue, adjacent normal mucosal tissue, and peripheral blood leukocytes revealed that the patient had germline Asp820Gly mutation in exon 17 of the KIT gene. This is the first case with germline Asp820Gly mutation in exon 17 of the KIT gene.


Asunto(s)
Neoplasias Gastrointestinales/patología , Tumores del Estroma Gastrointestinal/patología , Mutación de Línea Germinal , Proteínas Proto-Oncogénicas c-kit/genética , Anciano , Femenino , Neoplasias Gastrointestinales/genética , Tumores del Estroma Gastrointestinal/genética , Humanos , Pronóstico
18.
BMC Surg ; 20(1): 163, 2020 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-32698798

RESUMEN

BACKGROUND: Delta-shaped anastomosis is a common method of intracorporeal gastroduodenostomy in totally laparoscopic distal gastrectomy. One common postoperative complication of this procedure is anastomotic stenosis, and endoscopic balloon dilatation is a major remedy for such complications. Other treatment strategies are necessary to manage unsuccessful endoscopic balloon dilatation. CASE PRESENTATION: We present a case where systemic steroid treatment was applied in sustained anastomotic stenosis after endoscopic balloon dilatation. We performed delta-shaped anastomosis in laparoscopic distal gastrectomy to treat early-stage gastric cancer in a patient. The patient experienced abdominal pain post-surgery; subsequent investigation revealed edematous anastomotic stenosis. The stenosis sustained even after endoscopic balloon dilatation and local steroid injection. Consequently, we applied systemic steroid treatment. CONCLUSION: Systemic steroid treatment improved the stenosis and no recurrence was observed. These results suggest that systemic steroid application could be useful to treat anastomotic stenosis.


Asunto(s)
Constricción Patológica/etiología , Gastrectomía/efectos adversos , Gastroenterostomía/efectos adversos , Glucocorticoides/administración & dosificación , Prednisolona/administración & dosificación , Neoplasias Gástricas , Administración Intravenosa , Anciano , Anastomosis Quirúrgica/efectos adversos , Anastomosis Quirúrgica/métodos , Constricción Patológica/tratamiento farmacológico , Dilatación/métodos , Edema/tratamiento farmacológico , Edema/etiología , Gastrectomía/métodos , Gastroenterostomía/métodos , Humanos , Laparoscopía , Masculino , Recurrencia Local de Neoplasia , Estudios Retrospectivos , Neoplasias Gástricas/cirugía
19.
J Lipid Res ; 59(10): 1906-1915, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30108153

RESUMEN

In pancreatic ß cells, ABCA1, a 254 kDa membrane protein, affects cholesterol homeostasis and insulin secretion. Angiotensin II, as the main effector of the renin-angiotensin system, decreases glucose-stimulated insulin secretion (GSIS). We examined the effect of angiotensin II on ABCA1 expression in primary pancreatic islets and INS-1 cells. Angiotensin II decreased ABCA1 protein and mRNA; angiotensin II type 1 receptor (AT1R) blockade rescued this ABCA1 repression. In parallel, angiotensin II suppressed the promoter activity of ABCA1, an effect that was abrogated by PD98095, a specific inhibitor of MAPK kinase (MEK). LXR enhanced ABCA1 promoter activity, and angiotensin II decreased the nuclear abundance of LXR protein. On a chromatin immunoprecipitation assay, LXR mediated the transcription of ABCA1 by directly binding to its promoter. Mutation of the LXR binding site on the ABCA1 promoter cancelled the effect of angiotensin II. Furthermore, angiotensin II induced cholesterol accumulation and impaired GSIS; inhibition of AT1R or MEK pathway reversed these effects. In summary, our study showed that angiotensin II suppressed ABCA1 expression in pancreatic islets and INS-1 cells, indicating that angiotensin II may influence GSIS by regulating ABCA1 expression. Additional research may address therapeutic needs in diseases such as diabetes mellitus.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Angiotensina II/farmacología , Colesterol/metabolismo , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Animales , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células Secretoras de Insulina/citología , Receptores X del Hígado/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratas , Transcripción Genética/efectos de los fármacos
20.
Am J Physiol Endocrinol Metab ; 315(6): E1232-E1241, 2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-30130150

RESUMEN

Recently, several clinical studies have suggested that adult growth hormone (GH) deficiency that also has low concentration of IGF1 is associated with an increased prevalence of fatty liver (FL). ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of lipid efflux from cells to apolipoproteins and plays an important role on formation of FL. In this study, we determined the effects of IGF1 on ABCA1 expression in GH-deficient mice to clarify its effects on FL. Western blotting, real-time PCR, and a luciferase assay were employed to examine the effect of IGF1. The binding of FoxO1 to the ABCA1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Cholesterol accumulation was analyzed by Oil Red O stain and cholesterol content measurement. We confirmed that IGF1 upregulated the ABCA1 expression. The activity of a reporter construct containing the ABCA1 promoter was induced by IGF1, and this effect was blocked by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Constitutively active Akt stimulated the ABCA1 promoter activity, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element abolished the effect of IGF1. A ChIP assay indicated that FoxO1 mediated IGF1 transcriptional activity by directly binding to the ABCA1 promoter region. For in vivo experiments, we used an inhibitor for the GH receptor (Pegvisomant) to reduce the IGF1 level. A high-fat diet induced FL in mice (C57BL/6J) given Pegvisomant treatment. IGF1 treatment stimulated ABCA1 expression to improve cholesterol accumulation in these mice. These results show that the PI3K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF1 stimulation that suppressed FL in GH-deficient mice.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Colesterol/metabolismo , Enanismo Hipofisario/metabolismo , Hígado Graso/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Hígado/metabolismo , Animales , Dieta Alta en Grasa , Proteína Forkhead Box O1/metabolismo , Células Hep G2 , Hormona de Crecimiento Humana/análogos & derivados , Hormona de Crecimiento Humana/farmacología , Humanos , Hígado/efectos de los fármacos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos
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