Nr0b1 is a negative regulator of Zscan4c in mouse embryonic stem cells.
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ABSTRACT:
SUBMITTER: Fujii S
PROVIDER: S-EPMC5390923 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Although the differentiation of pluripotent cells in embryonic stem cells (ESCs) is often associated with protein kinase-mediated signaling pathways and Tousled-like kinase 1 (Tlk1) is required for development in several species, the role of Tlk1 in ESC function remains unclear. Here, we used mouse ESCs to study the function of Tlk1 in pluripotent cells. The knockdown (KD)-based Tlk1-deficient cells showed that Tlk1 is not essential for ESC self-renewal in an undifferentiated state. However, Tlk1-KD cells formed irregularly shaped embryoid bodies and induced resistance to differentiation cues, indicating their failure to differentiate into an embryoid body. Consistent with their failure to differentiate, Tlk1-KD cells failed to downregulate the expression of undifferentiated cell markers including Oct4, Nanog, and Sox2 during differentiation, suggesting a negative role of Tlk1. Interestingly, Tlk1 overexpression sufficiently downregulated the expression of core pluripotency factors possibly irrespective of its kinase activity, thereby leading to a partial loss of self-renewal ability even in an undifferentiated state. Moreover, Tlk1 overexpression caused severe growth defects and G2/M phase arrest as well as apoptosis. Collectively, our data suggest that Tlk1 negatively regulates the expression of pluripotency factors, thereby contributing to the scheduled differentiation of mouse ESCs.
| S-EPMC5762884 | biostudies-literature
Project description:BACKGROUND:The regulatory role of long noncoding RNAs (lncRNAs) have been partially proved in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). METHODS:In the current study, we investigated mouse ESC (mESC) self-renewal, differentiation, and proliferation in vitro by knocking down a lncRNA, growth arrest specific 5 (Gas5). A series of related indicators were examined by cell counting kit-8 (CCK-8) assay, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), Western blot, alkaline phosphatase staining, propidium iodide (PI) staining, Annexin V staining, competition growth assay, immunofluorescence, and chromatin immunoprecipitation (ChIP)-qPCR. An in vivo teratoma formation assay was also performed to validate the in vitro results. qRT-PCR, fluorescence-activated cell sorting (FACS), alkaline phosphatase staining, and immunofluorescence were used to evaluate the role of Gas5 during mouse iPSC reprogramming. The regulatory axis of Dicer-miR291a-cMyc-Gas5 and the relationship between Gas5 and Tet/5hmC in mESCs was examined by qRT-PCR, Dot blot, and Western blot. RESULTS:We identified that Gas5 was required for self-renewal and pluripotency of mESCs and iPSCs. Gas5 formed a positive feedback network with a group of key pluripotent modulators (Sox2, Oct4, Nanog, Tcl1, Esrrb, and Tet1) in mESCs. Knockdown of Gas5 promoted endodermal differentiation of mESCs and impaired the efficiency of iPSC reprogramming. In addition, Gas5 was regulated by the Dicer-miR291a-cMyc axis and was involved in the DNA demethylation process in mESCs. CONCLUSIONS:Taken together, our results suggest that the lncRNA Gas5 plays an important role in modulating self-renewal and pluripotency of mESCs as well as iPSC reprogramming.
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Project description:A gene regulation network orchestrates processes ensuring the maintenance of cellular identity and genome integrity. Small RNAs generated by the RNAse III DICER have emerged as central players in this network. Moreover, deletion of Dicer in mice leads to early embryonic lethality. To better understand the underlying mechanisms leading to this phenotype, we generated Dicer-deficient mouse embryonic stem cells (mESCs). Their detailed characterization revealed an impaired differentiation potential, and incapacity to exit from the pluripotency state. We also observed a strong accumulation of LINE-1 (L1s) transcripts, which was translated at protein level and led to an increased L1s retrotransposition. Our findings reveal Dicer as a new essential player that sustains mESCs self-renewal and genome integrity by controlling L1s regulation.
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Project description:Embryonic Stem Cells (ESCs) are expected to show a stable euploid karyotype, but in the last decade (sub)chromosomal aberrations have been systematically described in these cell lines when maintained in vitro. Culture conditions and long-term culture have been traditionally proposed as possible factors involved in the acquisition of chromosomal abnormalities. Thus, we analyzed the chromosome constitution, the undifferentiated state and the functional pluripotency of three different mouse ESCs grown under the same culture conditions. Two cell lines were unstable from early passages, whereas the third one retained its chromosome integrity after long-term culture despite using enzymatic methods for cell disaggregation. Trisomy 8 and 11 were clonally selected in both unstable cell lines, which also showed a higher growth rate than our normal cell line and suffered morphological changes in colony shape with increasing passage number. Regardless of the length of culture or the chromosome instability, all cell lines preserved their differentiation potential. These results confirm that double trisomy 8 and 11 confers a growth advantage to the abnormal cells, but not at the expense of cell differentiation. The presence of chromosome instability, widely related to tumor development and cancer disease, highlights the risk of using pluripotent cells in regenerative medicine.
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Project description:BackgroundEmbryonic Sertoli cells (eSCs) play an important role in sex determination and in male gonad development which makes them a very useful cell type for therapeutic applications. However, the deriving mechanism of Sertoli cells has been unclear and challenging to create a large number of quality eSCs. Therefore, this study aimed to create the eSCs induced from mouse embryonic stem (mES) cells by regulating defined factors and to explore the relevant regulatory mechanism.MethodsSix inducing factors, Sry, Sox9, SF1, WT1, GATA4, and Dmrt1, were respectively transduced into mES cells by lentiviral infection according to the experimental design. The test groups were identified by development stage-specific markers, AMH, Emx2, SF1, and FasL, using flow cytometry. Induced eSCs were determined by FasL and AMH biomarkers under immunofluorescence, immunocytochemistry, and flow cytometry. Moreover, the pluripotency markers, gonad development-related markers, epithelial markers and mesenchymal markers in test groups were transcriptionally determined by qPCR.ResultsIn this study, the co-overexpression of all the six factors effectively produced a large population of eSCs from mES cells in 35 days of culturing. These eSCs were capable of forming tubular-like and ring-like structures with functional performance. The results of flow cytometry indicated that the upregulation of GATA4 and WT1 contributed to the growth of somatic cells in the coelomic epithelium regarded as the main progenitor cells of eSCs. Whereas, SF1 facilitated the development of eSC precursor cells, and Sry and Sox9 promoted the determination of male development. Moreover, the overexpression of Dmrt1 was essential for the maintenance of eSCs and some of their specific surface biomarkers such as FasL. The cellular morphology, biomarker identification, and transcriptomic analysis aided in exploring the regulatory mechanism of deriving eSCs from mES cells.ConclusionConclusively, we have elucidated a differentiation roadmap of eSCs derived from mES cells with a relevant regulatory mechanism. Through co-overexpression of all these six factors, a large population of eSCs was successfully induced occupying 24% of the whole cell population (1 × 105 cells/cm2). By adopting this approach, a mass of embryonic Sertoli cells can be generated for the purpose of co-culture technique, organ transplantation, gonadal developmental and sex determination researches.
| S-EPMC6408820 | biostudies-literature
Project description:Mouse spermatogonial stem cells (mSSCs) may be reprogrammed to become pluripotent stem cells under in vitro culture conditions, due to epigenetic modifications, which are closely associated with the expression of transcription factors and epigenetic factors. Thus, this study was conducted to compare the gene expression of transcription factors and epigenetic factors in mSSCs and mouse embryonic stem cells (mESCs). Firstly, the freshly isolated mSSCs [mSSCs (f)] were enriched by magnetic-activated cell sorting with Thy1.2 (CD90.2) microbeads, and the typical morphological characteristics were maintained under in vitro culture conditions for over 5 months to form long-term propagated mSSCs [mSSCs (l)]. These mSSCs (l) expressed pluripotency?associated genes and were induced to differentiate into sperm. Our findings indicated that the mSSCs (l) expressed high levels of the transcription factors, Lin28 and Prmt5, and the epigenetic factors, Tet3, Parp1, Max, Tert and Trf1, in comparison with the mESCs, with the levels of Prmt5, Tet3, Parp1 and Tert significantly higher than those in the mESCs. There was no significant difference in Kdm2b expression between mSSCs (l) and mESCs. Furthermore, the gene expression of N-Myc, Dppa2, Tbx3, Nr5a2, Prmt5, Tet3, Parp1, Max, Tert and Trf1 in the mSSCs (l) was markedly higher in comparison to that in the mSSCs (f). Collectively, our results suggest that the mSSCs and the mESCs displayed differential gene expression profiles, and the mSSCs possessed the potential to acquire pluripotency based on the high expression of transcription factors and epigenetic factors. These data may provide novel insights into the reprogramming mechanism of mSSCs.
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Project description:Cell division is a process by which a mother cell divides into genetically identical sister cells, although sister cells often display considerable diversity. In this report, over 350 sister embryonic stem cells (ESCs) were isolated through a microdissection method, and then expression levels of 48 key genes were examined for each sister cell. Our system revealed considerable diversities between sister ESCs at both pluripotent and differentiated states, whereas the similarity between sister ESCs was significantly elevated in a 2i (MEK and GSK3b inhibitors) condition, which is believed to mimic the ground state of pluripotency. DNA methyltransferase 3a/3b were downregulated in 2i-grown ESCs, and the loss of DNA methyltransferases was sufficient to generate nearly identical sister cells. These results suggest that DNA methylation is a major cause of the diversity between sister cells at the pluripotent states, and thus demethylation per se plays an important role in promoting ESC's self-renewal.
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Project description:The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.
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Project description:Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.
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Project description:Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells.
| S-EPMC3343110 | biostudies-literature