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Pleiotropy of glycogen synthase kinase-3 inhibition by CHIR99021 promotes self-renewal of embryonic stem cells from refractory mouse strains - PubMed

Pleiotropy of glycogen synthase kinase-3 inhibition by CHIR99021 promotes self-renewal of embryonic stem cells from refractory mouse strains

Shoudong Ye et al. PLoS One. 2012.

Abstract

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. β-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 β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor-independent manner. Second, β-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. β-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 cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. LIF maintains B6 ES cell self-renewal in the presence of CHIR99021.

(A) Phase contrast image of B6 ES cells in the presence of 1000 U/ml LIF supplemented without or with 3 µM CHIR99021 (CH) for 4 days. Scale bars represent 100 µm. (B) Alkaline phosphatase staining of B6 ES cells in the presence of 1000 U/ml LIF supplemented without or with 3 µM CH for 4 days. Scale bars represent 100 µm. (C) Immunofluorescence staining for OCT4, NANOG, and KLF4 in B6 ES cells treated with 1000 U/ml LIF and 3 µM CH for 4 days. Cell nuclei were stained using Hoechst. Scale bars represent 100 µm. (D) Relative quantification of Oct4, Nanog, Rex1, Klf4, Tbx3, and Fgf5 mRNA in B6 ES cells in 1000 U/ml LIF culture without or with 3 µM CH, by qRT-PCR. Error bars represent the SD of three biological replicates. (E) B6 ES cells,1×104/well, cultured in different concentrations of LIF, ranging from 0 to 10,000 U/ml, in the absence or presence of 3 µM CH for 5 days, were assessed for alkaline phosphatase activity. Scale bars represent 100 µm.

Figure 2
Figure 2. Overexpression of β-catenin maintains B6 ESC self-renewal in the presence of LIF.

(A) Immunoblot analysis of phospho-β-catenin, β-catenin and β-actin levels in B6 ES cells cultured in 1000 U/ml LIF condition supplemented without or with 3 µM CH for 4 days. (B) Immunofluorescence analysis of 3 µM CH-treated and non-treated B6 ESC clones stained for β-catenin and Hoechst for nuclei in the presence of LIF for 4 days. Scale bars represent 100 µm. (C) Quantitative RT-PCR analysis of Axin2, T and Cdx1 transcript levels in 3 µM CH-treated and control B6 ES cells. Error bars represent the SD of three biological replicates. (D) β-catenin S33A protein stably expressed in ES cells was detected by immunoblotting. (E) Phase contrast microscopy image and alkaline phosphatase staining of control and β-catenin S33A-transfected B6 ES cells in the presence of 1000 U/ml LIF for 4 days. Scale bars represent 100 µm. (F) Immunofluorescence staining for OCT4, NANOG, and KLF4 in mock and β-catenin S33A-transfected B6 ES cells in the presence of 1000 U/ml LIF for 4 days. Cell nuclei were stained with Hoechst. Scale bars represent 100 µm. (G) Quantitative RT-PCR analysis of Oct4, Nanog, Rex1, Klf4, Tbx3, and Fgf5 transcript levels in mock and β-catenin S33A-transfected B6 ES cells in the presence of 1000 U/ml LIF for 4 days. Error bars represent the SD of three biological replicates. (H) β-catenin S33AΔC, β-catenin S33A and TCF4ΔN protein were detected by immunoblotting with anti-flag tag antibody from extracts of ES cells transfected with PiggyBac plasmids. Quantitative RT-PCR analysis of Axin2, T and Cdx1 transcript levels in β-catenin S33AΔC, β-catenin S33A, TCF4ΔN, and mock-transfected B6 ES cells. Error bars represent the SD of three biological replicates. (I) Alkaline phosphatase staining of mock-transfected, and β-cateninΔC over-expressing B6 ES cells cultured in LIF for 5 passages, and mock-transfected and TCF4ΔN-transfected B6 ESCs cultured in CHIR99021 plus LIF for 5 passages. Scale bars represent 100 µm.

Figure 3
Figure 3. Abrogating E-cadherin does not abolish self-renewal of undifferentiated B6 ESCs.

(A) Immunofluorescence staining of B6 ES cells for E-cadherin and cell nuclei in the presence of 1000 U/ml LIF supplemented without or with 3 µM CH for 4 days. Scale bars represent 100 µm. (B) Western blot analysis of E-Cadherin expression in B6 ES cells stably expressing a shRNA against E-Cadherin (Cdh1). Alkaline phosphatase staining of mock and Cdh1 shRNA-expressing B6 ES cells in the presence of 1000 U/ml LIF plus 3 µM CH at passage 5. Scale bars represent 100 µm. (C) Immunofluorescence staining for OCT4 and NANOG in mock and Cdh1 shRNA transfected B6 ES cells in the presence of 1000 U/ml LIF plus 3 µM CH at passage 5. Cell nuclei were detected by Hoechst staining. Scale bars represent 100 µm. (D) Western blot analysis of E-Cadherin expression in B6 ES cells treated with 1000 U/ml LIF or a combination of 1000 U/ml LIF and 3 µM CH for 4 days. β-actin was used as a loading control. (E) Western blot of lysates from B6 ES cells expressing Flag and Flag-tagged β-catenin S33A, S33A/Y654F, and S33A/Y654E mutants for E-cadherin after immunoprecipitation of cell lysates with Flag specific antibodies. (F) Alkaline phosphatase staining and quantitative analysis of alkaline phosphatase staining for Flag and Flag-tagged wild-type and Y654F and Y654E β-catenin mutants expressed in B6 cells in the presence of 1000 U/ml LIF. Scale bars represent 100 µm, and n>100 for each condition.

Figure 4
Figure 4. Forced c-Myc expression is beneficial to the self-renewal of B6 ES cells.

(A) Western blot analysis of c-Myc protein stability following inhibition of protein synthesis with CHX (50 µg/ml) for 1.5 and 3 hr in the presence or absence of 3 µM CH. (B) Western blot analysis of phospho-c-Myc and c-Myc expression. mRNA expression levels of c-Myc in B6 ES cells were determined by qRT-PCR in 1000 U/ml LIF supplemented without or with 3 µM CH at passage 3. Error bars represent the SD of three biological replicates. (C) c-Myc-ER fusion protein levels determined by western blotting. (D) ER and c-MycT58A-ER transfected B6 ES cells were cultured in 1000 U/ml LIF medium in the absence or presence of 1 µM 4OHT for 10 passages, and assessed by morphology (phase contrast) and alkaline phosphatase staining. Scale bars represent 100 µm. (E) Immunofluorescence staining for OCT4, NANOG, KLF4 and SSEA1 in ER and c-MycT58A-ER transfected B6 ES cells in 1000 U/ml LIF medium in the absence or presence of 1 µM 4OHT at passage 10. Scale bars represent 50 µm. (F) Quantitative RT-PCR analysis of Oct4, Nanog, Rex1, Klf4, Tbx3, and Fgf5 transcript levels in c-MycT58A-ER transfected B6 ES cells in the presence of 1000 U/ml LIF in the absence or presence of 1 µM 4OHT at passage 10. Error bars represent the SD of three biological replicates.

Figure 5
Figure 5. Both c-Myc, depending on its transformation activity, and N-myc can help to maintain self-renewal of B6 ES cells.

(A) ER and mutants c-Myc T58A, W136E, V394D, and L420P-ER induced transformation of NIH 3T3 cells in the presence of 1 µM 4OHT for one week. Scale bars represent 100 µm. (B) ER and mutants c-Myc T58A, W136E, V394D, and L420P-ER transfected B6 ES cells growing in 1000 U/ml LIF medium with or without 1 µM 4OHT were assayed for alkaline phosphatase staining at passage 5. Scale bars represent 100 µm. (C) Quantitative RT-PCR analysis of overexpressed N-Myc and L-Myc transcript levels in B6 ES cells. (D) Alkaline phosphatase staining of B6 ES cells transfected with N-Myc-ER, L-Myc-ER in 1000 U/ml LIF medium in the absence or presence of 1 µM 4OHT at passage 5. Error bars represent the SD of three biological replicates. Scale bars represent 50 µm.

Figure 6
Figure 6. LIF and CHIR99021 support derivation and maintenance of ES cells from BALB/c mouse.

(A, B) Phase contrast image and alkaline phosphatase staining of BALB/c ES cells in 1000 U/ml LIF medium supplemented without or with 3 µM CH for 4 days. Scale bars represent 100 µm. (C) Immunofluorescence staining of OCT4 and NANOG in BALB/c ES cells in 1000 U/ml LIF medium supplemented without or with 3 µM CH for 5 days. Nuclei were stained with Hoechst. Scale bars represent 100 µm. (D) Alkaline phosphatase staining of MycT58A-transfected BALB/c ES cells in 1000 U/ml LIF medium supplemented without or with 1 µM 4OHT for 4 days. Scale bars represent 100 µm. (E) Immunofluorescence staining of OCT4 and NANOG in MycT58A-ER-transfected BALB/c ES cells in 1000 U/ml LIF medium supplemented without or with 1 µM 4OHT for 4 days. Nuclei were stained with Hoechst. Scale bars represent 100 µm. (F) Alkaline phosphatase staining of β-catenin-transfected BALB/c ES cells in 1000 U/ml LIF medium. Scale bars represent 100 µm. (G) Immunofluorescence staining for OCT4 and NANOG in β-catenin transfected BALB/c ES cells in 1000 U/ml LIF medium. Nuclei were stained with Hoechst. Scale bars represent 100 µm.

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References

    1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154–156. - PubMed
    1. Garland LG, Marrion NV, Martin GR. The extraneuronal O-methylation of 3H-(+)isoprenaline by guinea-pig tracheal rings in vitro. Naunyn Schmiedebergs Arch Pharmacol. 1981;318:88–93. - PubMed
    1. Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci U S A. 1997;94:5709–5712. - PMC - PubMed
    1. Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988;336:688–690. - PubMed
    1. Niwa H. How is pluripotency determined and maintained? Development. 2007;134:635–646. - PubMed

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