Differentiation of Mouse Embryonic Stem Cells after RNA Interference-Mediated Silencing of OCT4 and Nanog

Hough, Shelley R.; Clements, Ian P.; Welch, Peter J.; Wiederholt, Kristin

doi:10.1634/stemcells.2005-0475

Cited by 133 publications

(120 citation statements)

References 26 publications

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“…3A). This finding resembled the parietal endoderm phenotype observed in Nanog KO ES cells (18) and that after knockdown of Nanog by chemically modified RNAi compounds (22). Quantitative real-time PCR (qRT-PCR) analysis of total RNA collected from induced, feeder-free Ainv15 shRNA-mir-Nanog cells indicated a reduction in stem cellspecific (Nanog, Rex1, and Dax1) gene expression, but a derepression of endoderm-specific (Gata6 and Bmp2) genes (Fig.…”

Section: Functional Consequences and Rescue Of Nanog Knockdown In Ainv15supporting

confidence: 78%

Site-directed, virus-free, and inducible RNAi in embryonic stem cells

Wang

Theunissen

Orkin

2007

Proc. Natl. Acad. Sci. U.S.A.

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RNAi is a powerful tool for interrogating gene function in ES cells.Combining the high penetrance of a microRNA-embedded shRNA (shRNA-mir) cassette with a locus-defined, inducible expression strategy, we developed a system for RNAi in mouse ES cells. An shRNA-mir cassette is targeted near the constitutively active HPRT locus under a tetracycline (tet)-regulatable promoter through Cremediated site-specific recombination. The major advantage of this system is that the shRNA-mir cassette can be targeted to a precise locus, allowing for control of shRNA-mir expression in an inducible fashion. Induction of an shRNA-mir directed against the pluripotency factor, Nanog, resulted in the loss of self-renewal and differentiation to parietal endoderm-like cells, which can be rescued by the introduction of an RNAi-immune version of Nanog cDNA. Knockdown efficiency can be enhanced by using multiple shRNA-mir hairpins against the target gene, which was further validated by knocking down two additional ES cell factors. This site-directed, virus-free, and tet-inducible RNAi system, designated as SDVFi RNAi in our study, presents an efficient option for controlled gene silencing in ES cells.microRNAs ͉ Nanog ͉ RNA interference ͉ tetracycline

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Section: Functional Consequences and Rescue Of Nanog Knockdown In Ainv15supporting

confidence: 78%

Site-directed, virus-free, and inducible RNAi in embryonic stem cells

Wang

Theunissen

Orkin

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The transcription factors oct-4 and nanog have essential roles in maintaining the pluripotency and self-renewal of both hESCs and mESCs (44 -46). Disruption of oct-4 or nanog results in the differentiation of both human and mouse embryonic stem cells (47)(48)(49). Currently, little is known about how these self-renewal genes are regulated.…”

Section: Discussionmentioning

confidence: 99%

“…Cells were cultured on irradiated mouse embryonic fibroblasts (MEFs) in hESCs culture medium consisting of 80% Dulbecco's modified Eagle's medium/F-12 (Invitrogen), 20% knock-out serum replacement (Invitrogen), 1 mM L-glutamine, 1% nonessential amino acids, 0.1 mM ␤-mercaptoethanol, and 4 ng/ml basic fibroblast growth factor (all from Invitrogen). Undifferentiated hESCs from passages 42-68 for H1 and passages [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] for H9 were used in this study. Cells were passaged with dispase (Invitrogen) every 5-7 days.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Apoptosis and Differentiation by p53 in Human Embryonic Stem Cells

Han

Qing

et al. 2007

Journal of Biological Chemistry

231

233

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The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs), hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However, despite the accumulation of p53, it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types, including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation.Human embryonic stem cells (hESCs) 3 are capable of essentially unlimited self-renewal and retain the developmental potential to differentiate into almost any cell type. These characteristics of hESCs make them attractive for tissue and cellbased therapies (1, 2). Previously, basic fibroblast growth factor and activin A were identified as self-renewal factors (3-6). However, for reasons that are not clear, hESCs often display high rates of spontaneous apoptosis and differentiation in culture, thus making the process of expanding these cells highly inefficient (3, 7-10). For example, Dravid et al. (8) reported that, under routine culture conditions, Ͼ30% of hESCs undergo spontaneous apoptosis. Furthermore, Ezashi et al. (12) showed that nearly 40% of hESCs undergo spontaneous differentiation after 12 days of culture in normoxic conditions. Finally, Maitra et al. (13) reported that multiple passages of hESCs can cause genomic alterations, which may limit the therapeutic application of hESCs. In contrast to hESCs, mouse embryonic stem cells (mESCs) undergo lower rates of spontaneous apoptosis and differentiation (14). Moreover, they maintain their pluripotency and genomic stability longer than hESCs (15). The reason for these different species-specific phenotypes in embryonic stem cells is currently unknown.The p53 tumor suppressor gene is a strong candidate for playing a role in the observed phenotypes of hESCs, because it regulates various cellular processes, including apoptosis, differentiation, and genomic integrity (16). In many cell types p53 plays a crucial role in controlling apoptosis and cell cycle arrest when these cells are exposed to stress-inducing conditions (17). In response to stress, p53 accumulates and transactivates downstream target genes such as mdm2 (responsible for the feedback degradation circuitry of p53), p21 (responsible for cell cycle control), bax, noxa, and puma (responsible for DNA damage-induced ...

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“…Nanog was discovered in embryonic stem cells (ESCs) for its ability to maintain ESCs self-renewal under conditions that would promote differentiation [2][3][4] . Accordingly, downregulation of Nanog in both mouse and human ESCs favours differentiation 1,5,6 . NANOG exerts its role in pluripotency in tight cooperation with two other pluripotency transcription factors, OCT4 (also known as POU5F1) and SOX2.…”

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confidence: 99%

Lineage-restricted function of the pluripotency factor NANOG in stratified epithelia

et al. 2014

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NANOG is a pluripotency transcription factor in embryonic stem cells; however, its role in adult tissues remains largely unexplored. Here we show that mouse NANOG is selectively expressed in stratified epithelia, most notably in the oesophagus where the Nanog promoter is hypomethylated. Interestingly, inducible ubiquitous overexpression of NANOG in mice causes hyperplasia selectively in the oesophagus, in association with increased cell proliferation. NANOG transcriptionally activates the mitotic programme, including Aurora A kinase (Aurka), in stratified epithelia, and endogenous NANOG directly binds to the Aurka promoter in primary keratinocytes. Interestingly, overexpression of Nanog or Aurka in mice increased proliferation and aneuploidy in the oesophageal basal epithelium. Finally, inactivation of NANOG in cell lines from oesophageal or head and neck squamous cell carcinomas (ESCCs or HNSCCs, respectively) results in lower levels of AURKA and decreased proliferation, and NANOG and AURKA expression are positively correlated in HNSCCs. Together, these results indicate that NANOG has a lineage-restricted mitogenic function in stratified epithelia.

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Differentiation of Mouse Embryonic Stem Cells after RNA Interference-Mediated Silencing of OCT4 and Nanog

Cited by 133 publications

References 26 publications

Site-directed, virus-free, and inducible RNAi in embryonic stem cells

Site-directed, virus-free, and inducible RNAi in embryonic stem cells

Regulation of Apoptosis and Differentiation by p53 in Human Embryonic Stem Cells

Lineage-restricted function of the pluripotency factor NANOG in stratified epithelia

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