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Musashi2 is required for the self-renewal and pluripotency of embryonic stem cells.

ABSTRACT: Recent studies have shown that the RNA binding protein Musashi 2 (Msi2) plays important roles during development. Msi2 has also been shown to be elevated in several leukemias and its elevated expression has been linked with poorer prognosis in these cancers. Additionally, in embryonic stem cells (ESC) undergoing the early stages of differentiation, Msi2 has been shown to associate with the transcription factor Sox2, which is required for the self-renewal of ESC. These findings led us to examine the effects of Msi2 on the behavior of ESC. We determined that ESC express two isoforms of Msi2, the larger canonical isoform (isoform 1) and a shorter, splice-variant isoform (isoform 2). Using multiple shRNA lentiviral vectors, we determined that knockdown of Msi2 disrupts the self-renewal of ESC and promotes their differentiation into cells that express markers associated with mesoderm, ectoderm, and trophectoderm. Moreover, our studies indicate that the extent of differentiation and the loss of self-renewal capacity correlate with the levels to which Msi2 levels were decreased. We extended these findings by engineering ESC to inducibly express either Msi2 isoform1 or isoform 2. We determined that ectopic expression of Msi2 isoform 1, but not isoform 2, enhances the cloning efficiency of ESC. In addition, we examined how Msi2 isoform 1 and isoform 2 affect the differentiation of ESC. Interestingly, ectopic expression of either Msi2 isoform 1 or isoform 2 does not affect the pattern of differentiation induced by retinoic acid. Finally, we show that ectopic expression of either isoform 1 or isoform 2 is not sufficient to block the differentiation that results from the knockdown of both isoforms of Msi2. Thus, it appears that both isoforms of Msi2 are required for the self-renewal of ESC.

SUBMITTER: Wuebben EL

PROVIDER: S-EPMC3319613 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Musashi2 is required for the self-renewal and pluripotency of embryonic stem cells.

Wuebben Erin L EL Mallanna Sunil K SK Cox Jesse L JL Rizzino Angie A

PloS one 20120404 4

Recent studies have shown that the RNA binding protein Musashi 2 (Msi2) plays important roles during development. Msi2 has also been shown to be elevated in several leukemias and its elevated expression has been linked with poorer prognosis in these cancers. Additionally, in embryonic stem cells (ESC) undergoing the early stages of differentiation, Msi2 has been shown to associate with the transcription factor Sox2, which is required for the self-renewal of ESC. These findings led us to examine ...[more]

PMID: 22496868

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Project description:BackgroundSelf-renewal and pluripotency are considered as unwavering features of embryonic stem cells (ESCs). How ESCs regulate the self-renewal and differentiation is a central question in development and regenerative medicine research. Epidermal growth factor receptor (EGFR) was identified as a critical regulator in embryonic development, but its role in the maintenance of ESCs is poorly understood.MethodsHere, EGFR was disrupted by its specific inhibitor AG1478 in mouse ESCs (mESCs), and its self-renewal and pluripotency were characterized according to their proliferation, expression of pluripotency markers, embryoid body (EB) formation, and mRNA expression patterns. We also used another EGFR inhibitor (gefitinib) and RNA interference assay to rule out the possibility of non-specific effects of AG1478.ResultsEGFR inhibition by AG1478 treatment in mESCs markedly reduced cell proliferation, caused cell cycle arrest at G0/G1 phase, and altered protein expressions of the cell cycle regulatory genes (CDK2 (decreased 11.3%) and proliferating cell nuclear antigen (decreased 25.2%)). The immunoreactivities and protein expression of pluripotency factors (OCT4 (decreased 26.9%)) also dramatically decreased, while the differentiation related genes (GATA4 (increased 1.6-fold)) were up-regulated in mESCs after EGFR inhibition. Meanwhile, EGFR inhibition in mESCs disrupted EB formation, indicating its impaired pluripotency. Additionally, the effects observed by EGFR inhibition with another inhibitor gefitinib and siRNA were consistent with those observed by AG1478 treatment in mESCs. These effects were manifested in the decreased expression of proliferative and pluripotency-related genes and the increased expression of genes involved in differentiation. Moreover, RNA-seq analysis displayed that transcript profiling was changed significantly after EGFR inhibition by AG1478. A large number of differentially expressed genes were involved in cell cycle, apoptotic process, epigenetic modification, and metabolic process, which were related to self-renewal and pluripotency, confirming that EGFR deficiency impaired self-renewal and pluripotency in mESCs.ConclusionsTaken together, our results demonstrated the importance of EGFR in guarding the stemness of mESCs.

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Musashi2 is required for the self-renewal and pluripotency of embryonic stem cells.

Publications

Musashi2 is required for the self-renewal and pluripotency of embryonic stem cells.

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