Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 62
Filtrar
1.
Cell Stem Cell ; 31(6): 921-939.e17, 2024 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-38692273

RESUMEN

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.


Asunto(s)
Nefronas , Organoides , Animales , Organoides/citología , Organoides/metabolismo , Humanos , Nefronas/citología , Ratones , Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Podocitos/metabolismo , Podocitos/citología , Riñón/patología , Riñón Poliquístico Autosómico Dominante/patología , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Modelos Biológicos , Edición Génica
2.
Int J Mol Med ; 53(2)2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-38063256

RESUMEN

The Kv11.1 potassium channel encoded by the Kcnh2 gene is crucial in conducting the rapid delayed rectifier K+ current in cardiomyocytes. Homozygous mutation in Kcnh2 is embryonically lethal in humans and mice. However, the molecular signaling pathway of intrauterine fetal loss is unclear. The present study generated a Kcnh2 knockout rat based on edited rat embryonic stem cells (rESCs). Kcnh2 knockout was embryonic lethal on day 11.5 of development due to a heart configuration defect. Experiments with human embryonic heart single cells (6.5­7 weeks post­conception) suggested that potassium voltage­gated channel subfamily H member 2 (KCNH2) plays a crucial role in the development of compact cardiomyocytes. By contrast, apoptosis was found to be triggered in the homozygous embryos, which could be attributed to the failure of KCNH2 to form a complex with integrin ß1 that was essential for preventing the process of apoptosis via inhibition of forkhead box O3A. Destruction of the KCNH2/integrin ß1 complex reduced the phosphorylation level of AKT and deactivated the glycogen synthase kinase 3 ß (GSK­3ß)/ß­catenin pathway, which caused early developmental abnormalities in rats. The present work reveals a basic mechanism by which KCNH2 maintains intact embryonic heart development.


Asunto(s)
Canal de Potasio ERG1 , Cardiopatías Congénitas , Animales , Femenino , Humanos , Ratones , Embarazo , Ratas , Desarrollo Embrionario , Canal de Potasio ERG1/genética , Canal de Potasio ERG1/metabolismo , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Cardiopatías Congénitas/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Miocitos Cardíacos/metabolismo
3.
bioRxiv ; 2023 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-37293038

RESUMEN

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identifying novel genes associated with kidney development and disease. A rapid, efficient, and scalable organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.

4.
Med Image Anal ; 80: 102478, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35691144

RESUMEN

Breast Ultrasound (BUS) has proven to be an effective tool for the early detection of cancer in the breast. A lesion segmentation provides identification of the boundary, shape, and location of the target, and serves as a crucial step toward accurate diagnosis. Despite recent efforts in developing machine learning algorithms to automate this process, problems remain due to the blurry or occluded edges and highly irregular nodule shapes. Existing methods often produce over-smooth or inaccurate results, failing the need of identifying detailed boundary structures which are of clinical interest. To overcome these challenges, we propose a novel boundary-rendering framework that explicitly highlights the importance of boundary for automated nodule segmentation in BUS images. It utilizes a boundary selection module to automatically focuses on the ambiguous boundary region and a graph convolutional-based boundary rendering module to exploit global contour information. Furthermore, the proposed framework embeds nodule classification via semantic segmentation and encourages co-learning across tasks. Validation experiments were performed on different BUS datasets to verify the robustness of the proposed method. Results show that the proposed method outperforms states-of-art segmentation approaches (Dice=0.854, IOU=0.919, HD=17.8) in nodule delineation, as well as obtains a higher classification accuracy than classical classification models.


Asunto(s)
Algoritmos , Procesamiento de Imagen Asistido por Computador , Mama/diagnóstico por imagen , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Ultrasonografía , Ultrasonografía Mamaria/métodos
5.
Med Image Anal ; 80: 102490, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35717873

RESUMEN

Ultrasound (US) plays a vital role in breast cancer screening, especially for women with dense breasts. Common practice requires a sonographer to recognize key diagnostic features of a lesion and record a single or several representative frames during the dynamic scanning before performing the diagnosis. However, existing computer-aided diagnosis tools often focus on the final diagnosis process while neglecting the influence of the keyframe selection. Moreover, the lesions could have highly-irregular shapes, varying sizes, and locations during the scanning. The recognition of diagnostic characteristics associated with the lesions is challenging and also faces severe class imbalance. To address these, we proposed a reinforcement learning-based framework that can automatically extract keyframes from breast US videos of unfixed length. It is equipped with a detection-based nodule filtering module and a novel reward mechanism that can integrate anatomical and diagnostic features of the lesions into keyframe searching. A simple yet effective loss function was also designed to alleviate the class imbalance issue. Extensive experiments illustrate that the proposed framework can benefit from both innovations and is able to generate representative keyframe sequences in various screening conditions.


Asunto(s)
Neoplasias de la Mama , Ultrasonografía Mamaria , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Diagnóstico por Computador , Detección Precoz del Cáncer , Femenino , Humanos
7.
Biochem Biophys Res Commun ; 571: 8-13, 2021 09 24.
Artículo en Inglés | MEDLINE | ID: mdl-34298338

RESUMEN

Rats have long been an ideal model for disease research in the field of biomedicine, but the bottleneck of in vitro culture of rat embryonic stem (ES) cells hindered the wide application as genetic disease models. Here, we optimized a special medium which we named 5N-medium for rat embryonic stem cells, which improved the in vitro cells with better morphology and higher pluripotency. We then established a drug selection schedule harboring a prior selection of 12 h that achieved a higher positive selection ratio. These treatments induced at least 50% increase of homologous recombination efficiency compared with conventional 2i culture condition. Moreover, the ratio of euploid ES clones also increased by 50% with a higher germline transmission rate. Finally, we successfully knocked in a 175 kb human Bacterial Artificial Chromosome (BAC) fragment to rat ES genome through recombinase mediated cassette exchange (RMCE). Hence, we provide a promising system for generating sophisticated rat models which could be benefit for biomedical researches.


Asunto(s)
Células Madre Embrionarias/citología , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley
8.
Stem Cells ; 39(4): 443-457, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33426760

RESUMEN

ERG1, a potassium ion channel, is essential for cardiac action potential repolarization phase. However, the role of ERG1 for normal development of the heart is poorly understood. Using the rat embryonic stem cells (rESCs) model, we show that ERG1 is crucial in cardiomyocyte lineage commitment via interactions with Integrin ß1. In the mesoderm phase of rESCs, the interaction of ERG1 with Integrin ß1 can activate the AKT pathway by recruiting and phosphorylating PI3K p85 and focal adhesion kinase (FAK) to further phosphorylate AKT. Activation of AKT pathway promotes cardiomyocyte differentiation through two different mechanisms, (a) through phosphorylation of GSK3ß to upregulate the expression levels of ß-catenin and Gata4; (b) through promotion of nuclear translocation of nuclear factor-κB by phosphorylating IKKß to inhibit cell apoptosis, which occurs due to increased Bcl2 expression. Our study provides solid evidence for a novel role of ERG1 on differentiation of rESCs into cardiomyocytes.


Asunto(s)
Canal de Potasio ERG1/genética , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Integrina beta1/genética , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Animales , Apoptosis/genética , Diferenciación Celular , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Canal de Potasio ERG1/metabolismo , Embrión de Mamíferos , Células Madre Embrionarias/citología , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Integrina beta1/metabolismo , Miocitos Cardíacos/citología , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Transducción de Señal , beta Catenina/genética , beta Catenina/metabolismo
9.
Int J Stem Cells ; 13(2): 192-201, 2020 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-32587136

RESUMEN

BACKGROUND AND OBJECTIVES: Tcfs and Lef1 are DNA-binding transcriptional factors in the canonical Wnt signaling pathway. In the absence of ß-catenin, Tcfs and Lef1 generally act as transcriptional repressors with co-repressor proteins such as Groucho, CtBP, and HIC-5. However, Tcfs and Lef1 turn into transcriptional activators during the interaction with ß-catenin. Therefore, the activity of Tcfs and Lef1 is regulated by ß-catenin. However, the intrinsic role of Tcfs and Lef1 has yet to be examined. The purpose of this study was to determine whether Tcfs and Lef1 play differential roles in the regulation of self-renewal and differentiation of mouse ES cells. METHODS AND RESULTS: Interestingly, the expression of Tcfs and Lef1 was dynamically altered under various differentiation conditions, such as removal of LIF, EB formation and neuronal differentiation in N2B27 media, suggesting that the function of each Tcf and Lef1 may vary in ES cells. Ectopic expression of Tcf1 or the dominant negative form of Lef1 (Lef1-DN) contributes to ES cells to self-renew in the absence of leukemia inhibitory factor (LIF), whereas ectopic expression of Tcf3, Lef1 or Tcf1-DN did not support ES cells to self-renew. Ectopic expression of either Lef1 or Lef1-DN blocked neuronal differentiation, suggesting that the transient induction of Lef1 was necessary for the initiation and progress of differentiation. ChIP analysis shows that Tcf1 bound to Nanog promoter and ectopic expression of Tcf1 enhanced the transcription of Nanog. CONCLUSIONS: The overall data suggest that Tcf1 plays a critical role in the maintenance of stemness whereas Lef1 is involved in the initiation of differentiation.

10.
Biochim Biophys Acta Mol Cell Res ; 1866(6): 971-977, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30857870

RESUMEN

During embryonic development, neural stem cells (NSCs) emerge as early as the neural plate stage and give rise to the nervous system. Early-stage NSCs express Sry-related-HMG box-1 (Sox1) and are biased towards neuronal differentiation. However, long-term maintenance of early-stage NSCs in vitro remains a challenge. Here, we report development of a defined culture condition for the long-term maintenance of Sox1-positive early-stage mouse NSCs. The proliferative ability of these Sox1-positive NSCs was confirmed by clonal propagation. Compared to the NSCs cultured using the traditional culture condition, the long-term self-renewing Sox1-positive NSCs efficiently differentiate into neurons and exhibit an identity representative of the anterior and midbrain regions. These early-stage Sox1-positive NSCs could also be switched to late-stage NSCs by being cultured with bFGF/EGF, which can then differentiate into astrocytes and oligodendrocytes. The long-term self-renewing Sox1-positive NSCs were defined as naïve NSCs, based on their high neuronal differentiation capacity and anterior regional identity. This culture condition provides a robust platform for further dissection of the NSC self-renewal mechanism and promotes potential applications of NSCs for cell-based therapy on nervous system disorders.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células-Madre Neurales/citología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Autorrenovación de las Células/efectos de los fármacos , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Ratones , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal/efectos de los fármacos
11.
J Cell Mol Med ; 23(5): 3629-3640, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30907509

RESUMEN

The generation of germline competent rat embryonic stem cells (rESCs) allows the study of their lineage commitment. Here, we developed a highly efficient system for rESC-derived cardiomyocytes, and even the formation of three-dimensional (3D)-like cell clusters with cTNT and α-Actinin. We have validated that laminin can interact with membrane integrin to promote the phosphorylation of both phosphatidylinositol 3-kinase (PI3K) p85 and the focal adhesion kinase (FAK). In parallel, GATA4 was up-regulated. Upon inhibiting the integrin, laminin loses the effect on cardiomyocyte differentiation, accompanied with a down-regulation of phosphorylation level of PI3K p85 and FAK. Meanwhile, the expression of Gata4 was inhibited as well. Taken together, laminin is a crucial component in the differentiation of rESCs into cardiomyocytes through increasing their proliferation via interacting with integrin pathway. These results provide new insights into the pathways mediated by extracellular laminin involved in the fate of rESC-derived cardiomyocytes.


Asunto(s)
Diferenciación Celular/fisiología , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Células Madre Embrionarias/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Integrinas/metabolismo , Laminina/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Expresión Génica/efectos de los fármacos , Laminina/farmacología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiología
12.
Hepatology ; 67(3): 1041-1055, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29024000

RESUMEN

Notch signaling plays an emerging role in the regulation of immune cell development and function during inflammatory response. Activation of the ras homolog gene family member A/Rho-associated protein kinase (ROCK) pathway promotes leukocyte accumulation in tissue injury. However, it remains unknown whether Notch signaling regulates ras homolog gene family member A/ROCK-mediated immune responses in liver ischemia and reperfusion (IR) injury. This study investigated intracellular signaling pathways regulated by Notch receptors in the IR-stressed liver and in vitro. In a mouse model of IR-induced liver inflammatory injury, we found that mice with myeloid-specific Notch1 knockout showed aggravated hepatocellular damage, with increased serum alanine aminotransferase levels, hepatocellular apoptosis, macrophage/neutrophil trafficking, and proinflammatory mediators compared to Notch1-proficient controls. Unlike in the controls, myeloid Notch1 ablation diminished hairy and enhancer of split-1 (Hes1) and augmented c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), JNK, ROCK1, and phosphatase and tensin homolog (PTEN) activation in ischemic livers. Disruption of JSAP1 in myeloid-specific Notch1 knockout livers improved hepatocellular function and reduced JNK, ROCK1, PTEN, and toll-like receptor 4 activation. Moreover, ROCK1 knockdown inhibited PTEN and promoted Akt, leading to depressed toll-like receptor 4. In parallel in vitro studies, transfection of lentivirus-expressing Notch1 intracellular domain promoted Hes1 and inhibited JSAP1 in lipopolysaccharide-stimulated bone marrow-derived macrophages. Hes1 deletion enhanced JSAP1/JNK activation, whereas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated JSAP1 knockout diminished ROCK1/PTEN and toll-like receptor 4 signaling. CONCLUSION: Myeloid Notch1 deficiency activates the ras homolog gene family member A/ROCK pathway and exacerbates hepatocellular injury by inhibiting transcriptional repressor Hes1 and inducing scaffold protein JSAP1 in IR-triggered liver inflammation; our findings underscore the crucial role of the Notch-Hes1 axis as a novel regulator of innate immunity-mediated inflammation and imply the therapeutic potential for the management of organ IR injury in transplant recipients. (Hepatology 2018;67:1041-1055).


Asunto(s)
Hígado/patología , Receptor Notch1/genética , Daño por Reperfusión/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Apoptosis/genética , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Hígado/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Especies Reactivas de Oxígeno , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Notch1/metabolismo , Transducción de Señal , Proteína de Unión al GTP rhoA
13.
Dev Cell ; 43(5): 563-576.e4, 2017 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-29207259

RESUMEN

Glycogen synthase kinase 3 (GSK3) plays a central role in diverse cellular processes. GSK3 has two mammalian isozymes, GSK3α and GSK3ß, whose functions remain ill-defined because of a lack of inhibitors that can distinguish between the two highly homologous isozymes. Here, we show that GSK3α and GSK3ß can be selectively inhibited in mouse embryonic stem cells (ESCs) using a chemical-genetic approach. Selective inhibition of GSK3ß is sufficient to maintain mouse ESC self-renewal, whereas GSK3α inhibition promotes mouse ESC differentiation toward neural lineages. Genome-wide transcriptional analysis reveals that GSK3α and GSK3ß have distinct sets of downstream targets. Furthermore, selective inhibition of individual GSK3 isozymes yields distinct phenotypes from gene deletion, highlighting the power of the chemical-genetic approach in dissecting kinase catalytic functions from the protein's scaffolding functions. Our study opens new avenues for defining GSK3 isozyme-specific functions in various cellular processes.


Asunto(s)
Diferenciación Celular/fisiología , Linaje de la Célula , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3/genética , Células Madre Embrionarias de Ratones/citología , Animales , Estudio de Asociación del Genoma Completo/métodos , Ratones , Ratones Noqueados , Fosforilación , Transducción de Señal/fisiología
14.
J Cell Sci ; 130(22): 3809-3817, 2017 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-28982712

RESUMEN

TFCP2L1 is a transcription factor that is crucial for self-renewal of mouse embryonic stem cells (mESCs). How TFCP2L1 maintains the pluripotent state of mESCs, however, remains unknown. Here, we show that knockdown of Tfcp2l1 in mESCs induces the expression of endoderm, mesoderm and trophectoderm markers. Functional analysis of mutant forms of TFCP2L1 revealed that TFCP2L1 depends on its N-terminus and CP2-like domain to maintain the undifferentiated state of mESCs. The N-terminus of TFCP2L1 is mainly associated with the suppression of mesoderm and trophectoderm differentiation, while the CP2-like domain is closely related to the suppression of endoderm commitment. Further studies showed that MTA1 directly interacts with TFCP2L1 and is indispensable for the TFCP2L1-mediated self-renewal-promoting effect and endoderm-inhibiting action. TFCP2L1-mediated suppression of mesoderm and trophectoderm differentiation, however, seems to be due to downregulation of Lef1 expression. Our study thus provides an expanded understanding of the function of TFCP2L1 and the pluripotency regulation network of ESCs.


Asunto(s)
Factor de Unión 1 al Potenciador Linfoide/metabolismo , Células Madre Embrionarias de Ratones/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Autorrenovación de las Células , Células Cultivadas , Ectodermo/citología , Mesodermo/citología , Ratones , Transactivadores
15.
Dev Biol ; 431(2): 272-281, 2017 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-28943339

RESUMEN

ß-catenin-mediated signaling has been extensively studied in regard to its role in the regulation of human embryonic stem cells (hESCs). However, the results are controversial and the mechanism by which ß-catenin regulates the hESC fate remains unclear. Here, we report that ß-catenin and γ-catenin are functionally redundant in mediating hESC adhesion and are required for embryoid body formation, but both genes are dispensable for hESC maintenance, as the undifferentiated state of ß-catenin and γ-catenin double deficient hESCs can be maintained. Overexpression of ß-catenin induces rapid hESC differentiation. Functional assays revealed that TCF1 plays a crucial role in hESC differentiation mediated by ß-catenin. Forced expression of TCF1, but not other LEF1/TCF family members, resulted in hESC differentiation towards the definitive endoderm. Conversely, knockdown of TCF1 or inhibition of the interaction between TCF1 and ß-catenin delayed hESC exit from pluripotency. Furthermore, we demonstrated that GATA6 plays a predominant role in TCF1-mediated hESC differentiation. Knockdown of GATA6 completely eliminated the effect of TCF1, while forced expression of GATA6 induced hESC differentiation. Our data thus reveal more detailed mechanisms for ß-catenin in regulating hESC fate decisions and will expand our understanding of the self-renewal and differentiation circuitry in hESCs.


Asunto(s)
Linaje de la Célula , Factor de Transcripción GATA6/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Transducción de Señal , beta Catenina/metabolismo , Adhesión Celular , Diferenciación Celular , Autorrenovación de las Células , Desmoplaquinas/metabolismo , Endodermo/citología , Humanos , Transcripción Genética , Regulación hacia Arriba , gamma Catenina
16.
PLoS One ; 12(9): e0185714, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28961274

RESUMEN

Activation of signal transducer and activator of transcription 3 (STAT3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal. Our previous study showed that trans-acting transcription factor 5 (Sp5), an LIF/STAT3 downstream target, supports mESC self-renewal. However, the mechanism by which Sp5 exerts these effects remains elusive. Here, we found that Nanog is a direct target of Sp5 and mediates the self-renewal-promoting effect of Sp5 in mESCs. Overexpression of Sp5 induced Nanog expression, while knockdown or knockout of Sp5 decreased the Nanog level. Moreover, chromatin immunoprecipitation (ChIP) assays showed that Sp5 directly bound to the Nanog promoter. Functional studies revealed that knockdown of Nanog eliminated the mESC self-renewal-promoting ability of Sp5. Finally, we demonstrated that the self-renewal-promoting function of Sp5 was largely dependent on its zinc finger domains. Taken together, our study provides unrecognized functions of Sp5 in mESCs and will expand our current understanding of the regulation of mESC pluripotency.


Asunto(s)
Células Madre Embrionarias/citología , Proteína Homeótica Nanog/metabolismo , Factores de Transcripción/fisiología , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Ratones , Ratones Noqueados , Factores de Transcripción/genética
17.
J Biol Chem ; 292(41): 17121-17128, 2017 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-28848051

RESUMEN

The transcription factor Gbx2 (gastrulation brain homeobox 2) is a direct target of the LIF/STAT3 signaling pathway, maintains mouse embryonic stem cell (mESC) self-renewal, and facilitates mouse epiblast stem cell (mEpiSC) reprogramming to naïve pluripotency. However, the mechanism by which Gbx2 mediates its effects on pluripotency remains unknown. Here, using an RNA-Seq approach, we identified Klf4 (Kruppel-like factor 4) as a direct target of Gbx2. Functional studies indicated that Klf4 mediates the self-renewal-promoting effects of Gbx2, because knockdown of Klf4 expression abrogated the ability of Gbx2 to maintain the undifferentiated state of mESCs. We also found that Gbx2 largely depends on Klf4 to reprogram mEpiSCs to a mESC-like state. In summary, our study has uncovered a mechanism by which Gbx2 maintains and induces naïve pluripotency. These findings expand our understanding of the pluripotency control network and may inform the development of culture conditions for improved ESC maintenance and differentiation.


Asunto(s)
Reprogramación Celular/fisiología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Células Madre Embrionarias de Ratones/citología
18.
Stem Cell Reports ; 9(3): 732-741, 2017 09 12.
Artículo en Inglés | MEDLINE | ID: mdl-28844657

RESUMEN

Mouse epiblast stem cells (mEpiSCs) and human embryonic stem cells (hESCs) are primed pluripotent stem cells whose self-renewal can be maintained through cytoplasmic stabilization and retention of ß-catenin. The underlying mechanism, however, remains largely unknown. Here, we show that cytoplasmic ß-catenin interacts with and retains TAZ, a Hippo pathway effector, in the cytoplasm. Cytoplasmic retention of TAZ promotes mEpiSC self-renewal in the absence of nuclear ß-catenin, whereas nuclear translocation of TAZ induces mEpiSC differentiation. TAZ is dispensable for naive mouse embryonic stem cell (mESC) self-renewal but required for the proper conversion of mESCs to mEpiSCs. The self-renewal of hESCs, like that of mEpiSCs, can also be maintained through the cytoplasmic retention of ß-catenin and TAZ. Our study indicates that how TAZ regulates cell fate depends on not only the cell type but also its subcellular localization.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular , Autorrenovación de las Células , Estratos Germinativos/citología , Ratones , Modelos Biológicos , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Unión Proteica , Transporte de Proteínas , Transactivadores , beta Catenina/metabolismo
19.
Stem Cell Reports ; 8(6): 1457-1464, 2017 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-28591647

RESUMEN

Embryonic stem cells (ESCs) are a unique tool for genetic perturbation of mammalian cellular and organismal processes additionally in humans offer unprecedented opportunities for disease modeling and cell therapy. Furthermore, ESCs are a powerful system for exploring the fundamental biology of pluripotency. Indeed understanding the control of self-renewal and differentiation is key to realizing the potential of ESCs. Building on previous observations, we found that mouse ESCs can be derived and maintained with high efficiency through insulation from differentiation cues combined with consolidation of an innate cell proliferation program. This finding of a pluripotent ground state has led to conceptual and practical advances, including the establishment of germline-competent ESCs from recalcitrant mouse strains and for the first time from the rat. Here, we summarize historical and recent progress in defining the signaling environment that supports self-renewal. We compare the contrasting requirements of two types of pluripotent stem cell, naive ESCs and primed post-implantation epiblast stem cells (EpiSCs), and consider the outstanding challenge of generating naive pluripotent stem cells from different mammals.


Asunto(s)
Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , Animales , Diferenciación Celular , Autorrenovación de las Células , Células Madre Embrionarias/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Factor Inhibidor de Leucemia/metabolismo , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
20.
Immunol Res ; 65(3): 739-749, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28286920

RESUMEN

Liver injury after experimental acetaminophen treatment is mediated both by direct hepatocyte injury through a P450-generated toxic metabolite and indirectly by activated liver Kupffer cells and neutrophils. This study was designed to investigate the role of Notch signaling in the regulation of innate immune responses in acetaminophen (APAP)-induced liver injury. Using a mouse model of APAP-induced liver injury, wild-type (WT) and toll-like receptor 4 knockout (TLR4 KO) mice were injected intraperitoneally with APAP or PBS. Some animals were injected with γ-secretase inhibitor DAPT or DMSO vehicle. For the in vitro study, bone marrow-derived macrophages (BMMs) were transfected with Notch1 siRNA, TLR4 siRNA, and non-specific (NS) siRNA and stimulated with LPS. Indeed, paracetamol/acetaminophen-induced liver damage was worse after Notch blockade with DAPT in wild-type mice, which was accompanied by significantly increased ALT levels, diminished hairy and enhancer of split-1 (Hes1), and phosphorylated Stat3 and Akt but enhanced high mobility group box 1 (HMGB1), TLR4, NF-κB, and NLRP3 activation after APAP challenge. Mice receiving DAPT increased macrophage and neutrophil accumulation and hepatocellular apoptosis. However, TLR4 KO mice that received DAPT reduced APAP-induced liver damage and NF-κB, NLRP3, and cleaved caspase-1 activation. BMMs transfected with Notch1 siRNA reduced Hes1 and phosphorylated Stat3 and Akt but augmented HMGB1, TLR4, NF-κB, and NLRP3. Furthermore, TLR4 siRNA knockdown resulted in decreased NF-κB and NLRP3 and cleaved caspase-1 and IL-1ß levels following LPS stimulation. These results demonstrate that Notch signaling regulates innate NLRP3 inflammasome activation through regulation of HMGB1/TLR4/NF-κB activation in APAP-induced liver injury. Our novel findings underscore the critical role of the Notch1-Hes1 signaling cascade in the regulation of innate immunity in APAP-triggered liver inflammation. This might imply a novel therapeutic potential for the drug-induced damage-associated lethal hepatitis.


Asunto(s)
Acetaminofén/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Hepatocitos/fisiología , Macrófagos/fisiología , Receptor Notch1/metabolismo , Factor de Transcripción HES-1/metabolismo , Acetaminofén/uso terapéutico , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Proteína HMGB1/metabolismo , Hepatocitos/efectos de los fármacos , Humanos , Inmunidad Innata , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , ARN Interferente Pequeño/genética , Receptor Notch1/genética , Transducción de Señal , Receptor Toll-Like 4/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...