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1.
Environ Toxicol ; 35(1): 66-77, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31507073

RESUMO

The heart is the first organ formed in the developing fetus, and abnormal development of the heart is a major cause of fetal death. The adverse effects of cigarette smoke on the heart have been well established, but it is not well understood how cigarette smoke components regulate signaling molecules and cardiac specific functions during the early differentiation stage of the embryonic heart. In this study, we identified changes in the size of mouse embryoid bodies (mEBs) in response to treatment with cigarette smoke extract (CSE) via regulation of HDAC2, p53, p21, and cyclin D1 protein expression, which are cardiac differentiation and cell-cycle markers, respectively. In addition, exposure of mouse embryonic stem cells (mESCs) to cigarette smoke components inhibited myocardial differentiation and development through the expression of HDAC1, HDAC2, GATA4, NKX2-5, TBX5, HAND1, and Troponin I. Long-term exposure studies showed that CSE and nicotine may delay the development of mouse cardiomyocytes from mESCs and inhibit the contractibility, which is a fundamental function of the heart. Taken together, these findings suggest that cigarette smoke components, including nicotine, may affect abnormal myocardial differentiation and development.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fumaça/efeitos adversos , Tabaco/toxicidade , Animais , Ciclo Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Histona Desacetilase 2/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fumar/efeitos adversos
2.
Iran Biomed J ; 24(1): 30-8, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31454861

RESUMO

Background: Germ cell development processes are influenced by soluble factors and intercellular signaling events between them and the neighboring somatic cells. More insight into the molecular biology of the germ cell development from embryonic stem (ES) cells and investigation of appropriate factors, specifically those targeting differentiation processes, is of great importance. In this study, we established an in vitro model with higher ES cell differentiation rate to germ cells, using adenylate cyclase activator, forskolin. Methods: ES cells were first cultured for five days, leading to embryoid body (EB) formation. Subsequently, the EB were dissociated and cultured for an additional three days in different forskolin concentrations of 5, 20, and 50 µM, with or without granulosa cells (GC) co-culture. On the 8th day, we analyzed the expressions of 5 germ cell-specific markers using quantitative real-time-PCR technique along with cell viability assay by MTT test. Results: Our results showed that in the GC-free cultures, a 50-µM concentration of forskolin resulted in a significant increase in Mvh, Gdf9, Scp3, and Rec8 expression levels in comparison to the control. However, when the cells were co-cultured with the GCs, 20-µM concentration of forskolin could also increase the expression of those germ cell-specific marker genes. Furthermore, results from the MTT assay showed enhanced cell proliferation and survival at all three concentrations of forskolin, but 20-µM concentration was the most potent one. Conclusion: These data indicate that forskolin can stimulate differentiation and proliferation, dose-dependently; however, the influence of GCs co-culturing should not go unnoticed.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Colforsina/farmacologia , Células da Granulosa/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Forma Celular/efeitos dos fármacos , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117438, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31377684

RESUMO

Raman microspectroscopy as a non-invasive and label-free technique was applied to diagnose the early stage differentiation of mouse embryonic stem cells. The differentiated and undifferentiated embryonic bodies (EBs) were cultured using handing drop method by the control of Leukemia Inhibitory Factor (LIF). Raman spectra of the periphery cells of differentiated EBs (PrE cells) and those of the interior of undifferentiated EBs (ES cells) were obtained to diagnose the stem cells of different differentiation. It was found from the spectra that the protein content increased as the cells differentiated. Principal component analysis (PCA) was carried out to further analyze the differences between ES cells and PrE cells. The first three principle components contained 98.19% from the total variance. Characteristic bands of ES and PrE cells were chosen to acquire Raman images of two cells according to the results of PCA. In the Raman images, PrE cells had a clear and bright outline in the peripheral areas while ES cells were difficult to identify, this could be a distinct characteristic to discriminate them. The result of the Raman images was consistent with the biological agreement that the differentiated cells were distributed around the periphery.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Murinas/citologia , Análise Espectral Raman/métodos , Animais , Células Cultivadas , Camundongos , Análise de Componente Principal
4.
PLoS Comput Biol ; 15(12): e1007351, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31877128

RESUMO

Identification of induced pluripotent stem (iPS) progenitor cells, the iPS forming cells in early stage of reprogramming, could provide valuable information for studying the origin and underlying mechanism of iPS cells. However, it is very difficult to identify experimentally since there are no biomarkers known for early progenitor cells, and only about 6 days after reprogramming initiation, iPS cells can be experimentally determined via fluorescent probes. What is more, the ratio of progenitor cells during early reprograming period is below 5%, which is too low to capture experimentally in the early stage. In this paper, we propose a novel computational approach for the identification of iPS progenitor cells based on machine learning and microscopic image analysis. Firstly, we record the reprogramming process using a live cell imaging system after 48 hours of infection with retroviruses expressing Oct4, Sox2 and Klf4, later iPS progenitor cells and normal murine embryonic fibroblasts (MEFs) within 3 to 5 days after infection are labeled by retrospectively tracing the time-lapse microscopic image. We then calculate 11 types of cell morphological and motion features such as area, speed, etc., and select best time windows for modeling and perform feature selection. Finally, a prediction model using XGBoost is built based on the selected six types of features and best time windows. Our model allows several missing values/frames in the sample datasets, thus it is applicable to a wide range of scenarios. Cross-validation, holdout validation and independent test experiments show that the minimum precision is above 52%, that is, the ratio of predicted progenitor cells within 3 to 5 days after viral infection is above 52%. The results also confirm that the morphology and motion pattern of iPS progenitor cells is different from that of normal MEFs, which helps with the machine learning methods for iPS progenitor cell identification.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Aprendizado de Máquina , Animais , Células Cultivadas , Reprogramação Celular , Biologia Computacional , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Imagem com Lapso de Tempo
5.
Cell Biochem Funct ; 37(8): 608-617, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31502671

RESUMO

Mouse embryonic stem cells (mESCs) are characterized by their self-renewal and pluripotency and are capable of differentiating into all three germ layers. For this reason, mESCs are considered a very important model for stem cell research and clinical applications in regenerative medicine. The pre-mRNA processing factor 4 (PRPF4) gene is known to have a major effect on pre-mRNA splicing and is also known to affect tissue differentiation during development. In this study, we investigated the effects of PRPF4 knockdown on mESCs. First, we allowed mESCs to differentiate naturally and observed a significant decrease in PRPF4 expression during the differentiation process. We then artificially induced the knockdown of PRPF4 in mESCs and observed the changes in the phenotype. When PRPF4 was knocked down, various genes involved in mESC pluripotency showed significantly decreased expression. In addition, mESC proliferation increased abnormally, accompanied by a significant increase in mESC colony size. The formation of mESC embryoid bodies and teratomas was delayed following PRPF4 knockdown. Based on these results, the reduced expression of PRPF4 affects mESC phenotypes and is a key factor in mESC. SIGNIFICANCE OF THE STUDY: Our results indicate that PRPF4 affects the properties of mESCs. Suppression of PRPF4 resulted in a decrease in pluripotency of mESC and promoted proliferation. In addition, suppression of PRPF4 also resulted in decreased apoptosis. Moreover, the inhibition of PRPF4 reduced the ability to differentiate and formation of teratoma in mESC. Our results demonstrated that PRPF4 is a key factor of controlling mESC abilities.


Assuntos
Diferenciação Celular , Proliferação de Células , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Animais , Células Cultivadas , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Teratoma/genética , Teratoma/patologia
6.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470631

RESUMO

Metal cations are associated with many biological processes. The effects of these cations on nucleic acids and chromatin were extensively studied in the early stages of nucleic acid and chromatin research. The results revealed that some monovalent and divalent metal cations, including Mg2+, profoundly affect the conformations and stabilities of nucleic acids, the folding of chromatin fibers, and the extent of chromosome condensation. Apart from these effects, there have only been a few reports on the functions of these cations. In 2007 and 2013, however, Mg2+-implicated novel phenomena were found: Mg2+ facilitates or enables both self-assembly of identical double-stranded (ds) DNA molecules and self-assembly of identical nucleosomes in vitro. These phenomena may be deeply implicated in the heterochromatin domain formation and chromatin-based phase separation. Furthermore, a recent study showed that elevation of the intranuclear Mg2+ concentration causes unusual differentiation of mouse ES (embryonic stem) cells. All of these phenomena seem to be closely related to one another. Mg2+ seems to be a key regulator of chromatin dynamics and chromatin-based biological processes.


Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Magnésio/farmacologia , Nucleossomos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Cromatina/química , Cromatina/metabolismo , Humanos , Magnésio/metabolismo , Camundongos , Conformação Molecular/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Nucleossomos/metabolismo
7.
Nat Commun ; 10(1): 4065, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492838

RESUMO

Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per-O-acetylated, which, however, can induce a long-overlooked side reaction, non-enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N-azidoacetylgalactosamine (GalNAz) as next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O-propionylated GalNAz (1,3-Pr2GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG.


Assuntos
Acetilglucosamina/metabolismo , Monossacarídeos/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Receptores Estrogênicos/metabolismo , Animais , Azidas/química , Azidas/metabolismo , Linhagem Celular Tumoral , Autorrenovação Celular , Células Cultivadas , Glicosilação , Células HeLa , Hexosaminas/metabolismo , Humanos , Células MCF-7 , Camundongos , Monossacarídeos/química , Células-Tronco Embrionárias Murinas/citologia , Células NIH 3T3 , Células-Tronco Pluripotentes/citologia , Processamento de Proteína Pós-Traducional
8.
Nat Methods ; 16(10): 991-993, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31384045

RESUMO

We report a molecular assay, Methyl-HiC, that can simultaneously capture the chromosome conformation and DNA methylome in a cell. Methyl-HiC reveals coordinated DNA methylation status between distal genomic segments that are in spatial proximity in the nucleus, and delineates heterogeneity of both the chromatin architecture and DNA methylome in a mixed population. It enables simultaneous characterization of cell-type-specific chromatin organization and epigenome in complex tissues.


Assuntos
Cromatina/metabolismo , Metilação de DNA , Análise de Célula Única/métodos , Animais , Ilhas de CpG , Conjuntos de Dados como Assunto , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo
9.
Nat Genet ; 51(9): 1356-1368, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406346

RESUMO

In mammalian cells, chromosomes are partitioned into megabase-sized topologically associating domains (TADs). TADs can be in either A (active) or B (inactive) subnuclear compartments, which exhibit early and late replication timing (RT), respectively. Here, we show that A/B compartments change coordinately with RT changes genome wide during mouse embryonic stem cell (mESC) differentiation. While A to B compartment changes and early to late RT changes were temporally inseparable, B to A changes clearly preceded late to early RT changes and transcriptional activation. Compartments changed primarily by boundary shifting, altering the compartmentalization of TADs facing the A/B compartment interface, which was conserved during reprogramming and confirmed in individual cells by single-cell Repli-seq. Differentiating mESCs altered single-cell Repli-seq profiles gradually but uniformly, transiently resembling RT profiles of epiblast-derived stem cells (EpiSCs), suggesting that A/B compartments might also change gradually but uniformly toward a primed pluripotent state. These results provide insights into how megabase-scale chromosome organization changes in individual cells during differentiation.


Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Cromossomos/genética , Replicação do DNA , Células-Tronco Embrionárias Murinas/metabolismo , Análise de Célula Única/métodos , Análise Espaço-Temporal , Animais , Diferenciação Celular , Núcleo Celular/genética , Células Cultivadas , Reprogramação Celular , Feminino , Genoma , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Neurônios/citologia , Neurônios/metabolismo
10.
Mol Cell ; 75(5): 905-920.e6, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31422875

RESUMO

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.


Assuntos
Diferenciação Celular/fisiologia , Metilação de DNA/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Células-Tronco Embrionárias Murinas/metabolismo , Transcrição Genética/fisiologia , Animais , Linhagem Celular , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
11.
Nat Commun ; 10(1): 2939, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270324

RESUMO

E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells.


Assuntos
Fator de Transcrição E2F4/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteína do Retinoblastoma/metabolismo , Ativação Transcricional , Animais , Ciclo Celular , Divisão Celular , Fator de Transcrição E2F4/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Família Multigênica , Proteína do Retinoblastoma/genética
12.
Nucleic Acids Res ; 47(17): 9069-9086, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350899

RESUMO

Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.


Assuntos
Cromatina/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Endoderma/citologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator 3-beta Nuclear de Hepatócito/genética , Código das Histonas , Histonas/metabolismo , Camundongos , Camundongos Knockout , Modelos Genéticos , Células-Tronco Embrionárias Murinas/citologia , Transdução de Sinais
13.
BMC Res Notes ; 12(1): 370, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262352

RESUMO

OBJECTIVE: Redox homeostasis maintenance is essential to bring about cellular functions. Particularly, embryonic stem cells (ESCs) have high fidelity mechanisms for DNA repair, high activity of different antioxidant enzymes and low levels of oxidative stress. Although the expression and activity of antioxidant enzymes are reduced throughout the differentiation, the knowledge about the transcriptional regulation of genes involved in defense against oxidative stress is yet restricted. Since glutathione is a central component of a complex system involved in preserving cellular redox status, we aimed to study whether the expression of the glutathione reductase (Gsr) gene, which encodes an essential enzyme for cellular redox homeostasis, is modulated by the transcription factors critical for self-renewal and pluripotency of ESCs. RESULTS: We found that Gsr gene is expressed in ESCs during the pluripotent state and it was upregulated when these cells were induced to differentiate, concomitantly with Nanog decreased expression. Moreover, we found an increase in Gsr mRNA levels when Nanog was downregulated by a specific shRNA targeting this transcription factor in ESCs. Our results suggest that Nanog represses Gsr gene expression in ESCs, evidencing a role of this crucial pluripotency transcription factor in preservation of redox homeostasis in stem cells.


Assuntos
Glutationa Redutase/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Homeobox Nanog/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica , Genes Reporter , Glutationa Redutase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/antagonistas & inibidores , Proteína Homeobox Nanog/metabolismo , Células-Tronco Pluripotentes/citologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
14.
Nucleic Acids Res ; 47(16): 8485-8501, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31304534

RESUMO

Endogenous retroviruses (ERVs) contribute to ∼10 percent of the mouse genome. They are often silenced in differentiated somatic cells but differentially expressed at various embryonic developmental stages. A minority of mouse embryonic stem cells (ESCs), like 2-cell cleavage embryos, highly express ERV MERVL. However, the role of ERVs and mechanism of their activation in these cells are still poorly understood. In this study, we investigated the regulation and function of the stage-specific expressed ERVs, with a particular focus on the totipotency marker MT2/MERVL. We show that the transcription factor Zscan4c functions as an activator of MT2/MERVL and 2-cell/4-cell embryo genes. Zinc finger domains of Zscan4c play an important role in this process. In addition, Zscan4c interacts with MT2 and regulates MT2-nearby 2-cell/4-cell genes through promoting enhancer activity of MT2. Furthermore, MT2 activation is accompanied by enhanced H3K4me1, H3K27ac, and H3K14ac deposition on MT2. Zscan4c also interacts with GBAF chromatin remodelling complex through SCAN domain to further activate MT2 enhancer activity. Taken together, we delineate a previously unrecognized regulatory axis that Zscan4c interacts with and activates MT2/MERVL loci and their nearby genes through epigenetic regulation.


Assuntos
Retrovirus Endógenos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Retroelementos , Fatores de Transcrição/genética , Animais , Cromatina/química , Cromatina/metabolismo , Embrião de Mamíferos , Retrovirus Endógenos/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Perfilação da Expressão Gênica , Ontologia Genética , Histonas/genética , Camundongos , Anotação de Sequência Molecular , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Fatores de Transcrição/metabolismo
15.
In Vitro Cell Dev Biol Anim ; 55(7): 473-481, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31214928

RESUMO

Leptin, a metabolic hormone, regulates the reproductive functions responding to both nutritional and body conditions. Embryonic stem cells play important roles in reproductive technology, but their derivation can be challenging. In this study, we evaluated the derivation rates of mouse embryonic stem cell (mESC) line from blastocysts developing in embryo culture media supplemented with different leptin concentrations. The results showed that addition of leptin into the embryo culture medium supported the in vitro development of mouse embryo. The mESC line derivation rates for media treated with 0, 10, 50, and 100 ng/ml of leptin were 61.24 % (54/88), 84.96 % (42/50), 81.79 % (61/76), and 85.78 % (56/67), respectively. In addition, leptin treatment of blastocysts upregulated the expression levels of the trophectoderm marker Cdx2, whereas inner cell mass markers Oct-4 and Nanog were not affected. mESC lines derived after leptin treatment demonstrated hallmarks of pluripotency, such as alkaline phosphatase activity, expression of, OCT4, NANOG, and SSEA1, as well as the ability to form embryoid bodies and well-differentiated teratomas. In conclusion, leptin has a positive effect on the derivation rate of mouse embryonic stem cell lines which may be, in part, due to its effects on the development of the trophectoderm cell lineage in the embryo.


Assuntos
Blastocisto/citologia , Proliferação de Células/efeitos dos fármacos , Leptina/farmacologia , Células-Tronco Embrionárias Murinas/citologia , Teratoma/metabolismo , Animais , Fator de Transcrição CDX2/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária , Corpos Embrioides/citologia , Antígenos CD15/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína Homeobox Nanog/biossíntese , Fator 3 de Transcrição de Octâmero/biossíntese , Teratoma/induzido quimicamente
16.
Dev Cell ; 50(2): 139-154.e5, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31204175

RESUMO

The epiblast is a pluripotent cell population first formed in preimplantation embryos, and its quality is important for proper development. Here, we examined the mechanisms of epiblast formation and found that the Hippo pathway transcription factor TEAD and its coactivator YAP regulate expression of pluripotency factors. After specification of the inner cell mass, YAP accumulates in the nuclei and activates TEAD. TEAD activity is required for strong expression of pluripotency factors and is variable in the forming epiblast. Cells showing low TEAD activity are eliminated from the epiblast through cell competition. Pluripotency factor expression and MYC control cell competition downstream of TEAD activity. Cell competition eliminates unspecified cells and is required for proper organization of the epiblast. These results suggest that induction of pluripotency factors by TEAD activity and elimination of unspecified cells via cell competition ensure the production of an epiblast with naive pluripotency.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Blastocisto/citologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/fisiologia , Camadas Germinativas/citologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Blastocisto/metabolismo , Proteínas de Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Feminino , Camadas Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo
17.
Nat Protoc ; 14(7): 1991-2014, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31160788

RESUMO

Ploidy represents the number of chromosome sets in a cell. Although gametes have a haploid genome (n), most mammalian cells have diploid genomes (2n). The diploid status of most cells correlates with the number of probable alleles for each autosomal gene and makes it difficult to target these genes via mutagenesis techniques. Here, we describe a 7-week protocol for the derivation of mouse haploid embryonic stem cells (hESCs) from female gametes that also outlines how to maintain the cells once derived. We detail additional procedures that can be used with cell lines obtained from the mouse Haplobank, a biobank of >100,000 individual mouse hESC lines with targeted mutations in 16,970 genes. hESCs can spontaneously diploidize and can be maintained in both haploid and diploid states. Mouse hESCs are genomically and karyotypically stable, are innately immortal and isogenic, and can be derived in an array of differentiated cell types; they are thus highly amenable to genetic screens and to defining molecular connectivity pathways.


Assuntos
Técnicas de Cultura de Células/métodos , Haploidia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Blastocisto/citologia , Linhagem Celular , Separação Celular/métodos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Fluxo de Trabalho
18.
Elife ; 82019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31157617

RESUMO

In amyotrophic lateral sclerosis (ALS) spinal motor neurons (SpMN) progressively degenerate while a subset of cranial motor neurons (CrMN) are spared until late stages of the disease. Using a rapid and efficient protocol to differentiate mouse embryonic stem cells (ESC) to SpMNs and CrMNs, we now report that ESC-derived CrMNs accumulate less human (h)SOD1 and insoluble p62 than SpMNs over time. ESC-derived CrMNs have higher proteasome activity to degrade misfolded proteins and are intrinsically more resistant to chemically-induced proteostatic stress than SpMNs. Chemical and genetic activation of the proteasome rescues SpMN sensitivity to proteostatic stress. In agreement, the hSOD1 G93A mouse model reveals that ALS-resistant CrMNs accumulate less insoluble hSOD1 and p62-containing inclusions than SpMNs. Primary-derived ALS-resistant CrMNs are also more resistant than SpMNs to proteostatic stress. Thus, an ESC-based platform has identified a superior capacity to maintain a healthy proteome as a possible mechanism to resist ALS-induced neurodegeneration.


Assuntos
Esclerose Amiotrófica Lateral/genética , Glicoproteínas de Membrana/genética , Neurônios Motores/metabolismo , Neurônios Eferentes/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Superóxido Dismutase-1/genética , Esclerose Amiotrófica Lateral/patologia , Esclerose Amiotrófica Lateral/terapia , Animais , Diferenciação Celular/genética , Nervos Cranianos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Neurônios Eferentes/efeitos dos fármacos , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/patologia
19.
Biochem J ; 476(11): 1585-1604, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31036718

RESUMO

Mitochondria play a central role in the maintenance of the naive state of embryonic stem cells. Many details of the mechanism remain to be fully elucidated. Solute carrier family 25 member 36 (Slc25a36) might regulate mitochondrial function through transporting pyrimidine nucleotides for mtDNA/RNA synthesis. Its physical role in this process remains unknown; however, Slc25a36 was recently found to be highly expressed in naive mouse embryonic stem cells (mESCs). Here, the function of Slc25a36 was characterized as a maintenance factor of mESCs pluripotency. Slc25a36 deficiency (via knockdown) has been demonstrated to result in mitochondrial dysfunction, which induces the differentiation of mESCs. The expression of key pluripotency markers (Pou5f1, Sox2, Nanog, and Utf1) decreased, while that of key TE genes (Cdx2, Gata3, and Hand1) increased. Cdx2-positive cells emerged in Slc25a36-deficient colonies under trophoblast stem cell culture conditions. As a result of Slc25a36 deficiency, mtDNA of knockdown cells declined, leading to impaired mitochondria with swollen morphology, decreased mitochondrial membrane potential, and low numbers. The key transcription regulators of mitochondrial biogenesis also decreased. These results indicate that mitochondrial dysfunction leads to an inability to support the pluripotency maintenance. Moreover, down-regulated glutathione metabolism and up-regulated focal adhesion reinforced and stabilized the process of differentiation by separately enhancing OCT4 degradation and promoting cell spread. This study improves the understanding of the function of Slc25a36, as well as the relationship of mitochondrial function with naive pluripotency maintenance and stem cell fate decision.


Assuntos
Glutationa/metabolismo , Proteínas Mitocondriais/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Animais , Fator de Transcrição CDX2/metabolismo , Diferenciação Celular/genética , Células Cultivadas , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Adesões Focais , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas de Transporte de Nucleotídeos/antagonistas & inibidores , Proteínas de Transporte de Nucleotídeos/genética , Fator 3 de Transcrição de Octâmero/metabolismo
20.
Nat Commun ; 10(1): 1931, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036804

RESUMO

Polycomb group (PcG) proteins play critical roles in the epigenetic inheritance of cell fate. The Polycomb Repressive Complexes PRC1 and PRC2 catalyse distinct chromatin modifications to enforce gene silencing, but how transcriptional repression is propagated through mitotic cell divisions remains a key unresolved question. Using reversible tethering of PcG proteins to ectopic sites in mouse embryonic stem cells, here we show that PRC1 can trigger transcriptional repression and Polycomb-dependent chromatin modifications. We find that canonical PRC1 (cPRC1), but not variant PRC1, maintains gene silencing through cell division upon reversal of tethering. Propagation of gene repression is sustained by cis-acting histone modifications, PRC2-mediated H3K27me3 and cPRC1-mediated H2AK119ub1, promoting a sequence-independent feedback mechanism for PcG protein recruitment. Thus, the distinct PRC1 complexes present in vertebrates can differentially regulate epigenetic maintenance of gene silencing, potentially enabling dynamic heritable responses to complex stimuli. Our findings reveal how PcG repression is potentially inherited in vertebrates.


Assuntos
Cromatina/metabolismo , Epigênese Genética , Inativação Gênica , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Cromatina/química , Retroalimentação Fisiológica , Histonas/genética , Histonas/metabolismo , Padrões de Herança , Camundongos , Mitose , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Transcrição Genética
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