Pleiotropy of Glycogen Synthase Kinase-3 Inhibition by CHIR99021 Promotes Self-Renewal of Embryonic Stem Cells from Refractory Mouse Strains

Background: Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. b-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. Methodology/Principal Findings: We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of b-catenin and c-Myc protein levels. Stabilized b-catenin promoted ES self-renewal through two mechanisms. First, b-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor–independent manner. Second, b-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. b-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. Conclusions/Significance: Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cell

New approach to ray-space interpolation for free viewpoint television

Ray-Space representation has superiority in rendering arbitrary viewpoint images of complicated scene in real-time. Ray-Space interpolation is one of the key techniques to make Ray-Space based Free Viewpoint Television (FTV) feasible. This paper presents a directionality based interpolation method for Ray-Space based FTV system, in which characteristic pixels are first extracted from sparse Ray-Space slice, and their directionalities are determined by block matching, while directionalities of other pixels to be interpolated are obtained by interpolating with the directionalities of these characteristic pixels. Experimental results show that the proposed method improves visual quality as well as PSNRs of rendered intermediate viewpoint image greatly.EI

Klf2 and Tfcp2l1, Two Wnt/β-Catenin Targets, Act Synergistically to Induce and Maintain Naive Pluripotency

Activation of Wnt/β-catenin signaling can induce both self-renewal and differentiation in naive pluripotent embryonic stem cells (ESCs). To gain insights into the mechanism by which Wnt/β-catenin regulates ESC fate, we screened and characterized its downstream targets. Here, we show that the self-renewal-promoting effect of Wnt/β-catenin signaling is mainly mediated by two of its downstream targets, Klf2 and Tfcp2l1. Forced expression of Klf2 and Tfcp2l1 can not only induce reprogramming of primed state pluripotency into naive state ESCs, but also is sufficient to maintain the naive pluripotent state of ESCs. Conversely, downregulation of Klf2 and Tfcp2l1 impairs ESC self-renewal mediated by Wnt/β-catenin signaling. Our study therefore establishes the pivotal role of Klf2 and Tfcp2l1 in mediating ESC self-renewal promoted by Wnt/β-catenin signaling

Depletion of Tcf3 and Lef1 maintains mouse embryonic stem cell self-renewal

Mouse and rat embryonic stem cell (ESC) self-renewal can be maintained by dual inhibition of glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK). Inhibition of GSK3 promotes ESC self-renewal by abrogating T-cell factor 3 (TCF3)-mediated repression of the pluripotency network. How inhibition of MEK mediates ESC self-renewal, however, remains largely unknown. Here, we show that inhibition of MEK can significantly suppress lymphoid enhancer factor 1 (LEF1) expression in mouse ESCs. Knockdown or knockout of Lef1 partially mimics the self-renewal-promoting effect of MEK inhibitors. Moreover, depletion of both Tcf3 and Lef1 enables maintenance of undifferentiated mouse ESCs without exogenous factors, cytokines or inhibitors. Transcriptome resequencing analysis reveals that LEF1 is closely associated with endoderm specification in ESCs. Thus, our study adds support to the notion that the key to maintaining the ESC ground state is to shield ESCs from differentiative cues

All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways

<p>Abstract</p> <p>Background</p> <p>All-trans retinoic acid (RA) is one of the most important morphogens with pleiotropic actions. Its embryonic distribution correlates with neural differentiation in the developing central nervous system. To explore the precise effects of RA on neural differentiation of mouse embryonic stem cells (ESCs), we detected expression of RA nuclear receptors and RA-metabolizing enzymes in mouse ESCs and investigated the roles of RA in adherent monolayer culture.</p> <p>Results</p> <p>Upon addition of RA, cell differentiation was directed rapidly and exclusively into the neural lineage. Conversely, pharmacological interference with RA signaling suppressed this neural differentiation. Inhibition of fibroblast growth factor (FGF) signaling did not suppress significantly neural differentiation in RA-treated cultures. Pharmacological interference with extracellular signal-regulated kinase (ERK) pathway or activation of Wnt pathway effectively blocked the RA-promoted neural specification. ERK phosphorylation was enhanced in RA-treated cultures at the early stage of differentiation.</p> <p>Conclusion</p> <p>RA can promote neural lineage entry by ESCs in adherent monolayer culture systems. This effect depends on RA signaling and its crosstalk with the ERK and Wnt pathways.</p

Sp5 induces the expression of Nanog to maintain mouse embryonic stem cell self-renewal

<div><p>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 <i>Sp5</i> induced <i>Nanog</i> expression, while knockdown or knockout of <i>Sp5</i> decreased the <i>Nanog</i> level. Moreover, chromatin immunoprecipitation (ChIP) assays showed that Sp5 directly bound to the Nanog promoter. Functional studies revealed that knockdown of <i>Nanog</i> 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.</p></div

Sp5 directly regulates the transcription of <i>Nanog</i>.

<p>(A) Independent validation of Nanog as an Sp5-bound target by ChIP-qPCR with fifteen primers set to scan different fragments of the Nanog promoter. Primers set at sites 1, 3, 5, 8, 9 and 15 represent significant enrichment. Data represent the mean±s.d. of three biological replicates. *p < 0.05, **p < 0.01 vs IgG. (B) Schematic illustration of luciferase reporter plasmids and <i>Sp5</i> expression activates the P_Nanog-luciferase reporter. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB.</p

Screening the downstream pluripotency genes regulated by <i>Sp5</i>.

<p>(A) HA-tagged <i>Sp5</i> was introduced into 46C mESCs and the protein level of HA-tagged <i>Sp5</i> was determined by Western blot. α-Tubulin was used as a loading control. (B) Phase-contrast and alkaline phosphatase (AP) staining images of PB and PB-<i>Sp5</i> mESCs cultured under serum-containing conditions in the absence of LIF for eight days. Scale bar, 100 μm. (C) Immunofluorescence of PB and PB-<i>Sp5</i> mESCs cultured under basal media conditions in the absence of LIF. Scale bar, 100 μm. (D) qRT–PCR analysis of the expression levels of mESC pluripotency markers (<i>Oct4</i>, <i>Sox2</i>, and <i>Nanog</i>) and differentiation-associated genes (<i>Gata4</i>, <i>Gata6</i>, and <i>T</i>) in PB and PB-<i>Sp5</i> 46C mESCs cultured in the absence of LIF. Data represent the mean±s.d of three biological replicates. **p < 0.01 vs PB. (E) qRT-PCR analysis of <i>Klf2/4/5</i>, <i>Nanog</i>, <i>Esrrb</i>, <i>Gbx2</i>, <i>Myc</i>, <i>Tfcp2l1</i>, <i>Tbx3</i> and <i>Pim1/3</i> expression in PB and PB-<i>Sp5</i> 46C mESCs cultured under LIF/serum-containing conditions. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB. (F) qRT–PCR analysis of <i>Sp5</i>, <i>Nanog</i> and <i>Klf2</i> transcripts in <i>scramble</i> and <i>Sp5</i> shRNA mESCs cultured under LIF/serum-containing conditions. Data represent the mean±s.d. of three biological replicates. *p < 0.05, **p < 0.01 vs <i>scramble</i> shRNA control.</p

Delineation of the zinc finger domains of Sp5 impairs the self-renewal-promoting effect of Sp5.

<p>(A) Schematic illustration of the <i>Sp5</i> deletion (Δ) mutants. Zinc finger domains are shown as green boxes. (B) Western blot analysis of Flag-tagged <i>Sp5</i> and <i>Sp5</i> deletion mutants with anti-FLAG antibody. (C) AP staining images of PB, PB-<i>Sp5</i> and <i>Sp5</i> deletion mutant mESCs cultured under serum-containing conditions in the absence of LIF for eight days. Scale bar, 100 μm. (D) Quantification of AP-positive colonies in Fig. 4C. (E) qRT–PCR analysis of the expression of <i>Nanog</i> in PB, PB-<i>Sp5</i> and <i>Sp5</i> deletion mutant mESCs cultured in LIF/serum-containing media. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB.</p