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Functional role of Mst1/Mst2 in embryonic stem cell differentiation.

Li P, Chen Y, Mak KK, Wong CK, Wang CC, Yuan P - PLoS ONE (2013)

Bottom Line: The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation.They also proliferate faster than wild type ES cells.Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Pathology, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China.

ABSTRACT
The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived Mst1/Mst2 double knockout (Mst-/-) ES cells to completely perturb Hippo signaling. We found that Mst-/- ES cells express higher level of Nanog than wild type ES cells and show differentiation resistance after LIF withdrawal. They also proliferate faster than wild type ES cells. Although Mst-/- ES cells can form embryoid bodies (EBs), their differentiation into tissues of three germ layers is distorted. Intriguingly, Mst-/- ES cells are unable to form teratoma. Mst-/- ES cells can differentiate into mesoderm lineage, but further differentiation to cardiac lineage cells is significantly affected. Microarray analysis revealed that ligands of non-canonical Wnt signaling, which is critical for cardiac progenitor specification, are significantly repressed in Mst-/- EBs. Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation.

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Related in: MedlinePlus

ES cell to cardiac progenitor cell differentiation is disturbed by Mst1/Mst2 depletion.(A) Heatmap of the expression of non-canonical Wnt signaling ligands (Wnt2, Wnt2b and Wnt5a) and canonical Wnt ligands (Wnt1, Wnt3a, Wnt8a and Wnt11) in day 4 and day 8 wild type EBs and Mst-/- EBs. (B) Relative mRNA levels of β-catenin in wild type and Mst-/- EBs at day 4 and day 8 during EB formation. Actin was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Immunoblotting analysis with antibodies against Active β-catenin and total β-catenin to check its expression in day 4 and day 8 wild type EBs and Mst-/- EBs. Gapdh1 was analyzed as an internal control. (D) Relative mRNA levels of Wnt5a during EB formation from day0 to day10. Actin was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (E) Recombinant Wnt5 were supplemented to the Mst-/-EB culture from day 2 and day 10. Wild type EBs and Mst-/- EBs were grown in non-Wnt5a supplemented medium as controls. The percentage of beating EBs was profiled on day 8 and day 10 after initiating EBs culture. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).
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pone-0079867-g007: ES cell to cardiac progenitor cell differentiation is disturbed by Mst1/Mst2 depletion.(A) Heatmap of the expression of non-canonical Wnt signaling ligands (Wnt2, Wnt2b and Wnt5a) and canonical Wnt ligands (Wnt1, Wnt3a, Wnt8a and Wnt11) in day 4 and day 8 wild type EBs and Mst-/- EBs. (B) Relative mRNA levels of β-catenin in wild type and Mst-/- EBs at day 4 and day 8 during EB formation. Actin was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Immunoblotting analysis with antibodies against Active β-catenin and total β-catenin to check its expression in day 4 and day 8 wild type EBs and Mst-/- EBs. Gapdh1 was analyzed as an internal control. (D) Relative mRNA levels of Wnt5a during EB formation from day0 to day10. Actin was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (E) Recombinant Wnt5 were supplemented to the Mst-/-EB culture from day 2 and day 10. Wild type EBs and Mst-/- EBs were grown in non-Wnt5a supplemented medium as controls. The percentage of beating EBs was profiled on day 8 and day 10 after initiating EBs culture. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).

Mentions: To check whether there is any link between the defect of Mst-/- ES cell cardiac lineage differentiation and the Wnt signaling abnormal regulation, we examined the expression of Wnt ligand genes in day 4 and day 8 wild type and Mst-/- EBs. The expression of canonical Wnt ligand gene Wnt1 and Wnt3a showed no change between in Mst-/- EBs and wild type EBs, while another canonical Wnt ligand gene Wnt8a was slightly increased (Figure 7A). RT-PCR and western blot to examine their downstream mediator β-catenin revealed that total and active β-catenin were at the similar level in wild type EBs and Mst-/- EBs, indicating the defect of Mst-/- ES cell to cardiac lineage differentiation may not be related to canonical Wnt signaling (Figure 7B and 7C). On the other hand, several non-canonical Wnt ligand genes, such as Wnt2, Wnt2b, and Wnt5a, were significantly downregulated in Mst-/- EBs (Figure 7A). Exogenous Wnt2 can enhance ES cell to cardiomyocyte differentiation. To validate whether Wnt5a can also enhance the differentiation, we first examined Wnt5a expression during wild type EB differentiation. We found that Wnt5a was dramatically increased in day 4 EBs (Figure 7D). Therefore we added Wnt5a recombinant protein to day 2 Mst-/- EB culture. Notably, Mst-/- EBs grown in medium supplemented with Wnt5a showed increase number of beating EBs compared to Mst-/- EBs grown in non-Wnt5a medium on day 8 and day 10, although less than wild type EBs, indicating that Wnt5a can partially rescue Mst-/- EB defect for cardiomyocyte differentiation (Figure 6B and 7E). Taken together, these data suggest that Mst1/Mst2 may involve in cardiomyocyte differentiation through crosstalking with non-canonical Wnt signaling.


Functional role of Mst1/Mst2 in embryonic stem cell differentiation.

Li P, Chen Y, Mak KK, Wong CK, Wang CC, Yuan P - PLoS ONE (2013)

ES cell to cardiac progenitor cell differentiation is disturbed by Mst1/Mst2 depletion.(A) Heatmap of the expression of non-canonical Wnt signaling ligands (Wnt2, Wnt2b and Wnt5a) and canonical Wnt ligands (Wnt1, Wnt3a, Wnt8a and Wnt11) in day 4 and day 8 wild type EBs and Mst-/- EBs. (B) Relative mRNA levels of β-catenin in wild type and Mst-/- EBs at day 4 and day 8 during EB formation. Actin was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Immunoblotting analysis with antibodies against Active β-catenin and total β-catenin to check its expression in day 4 and day 8 wild type EBs and Mst-/- EBs. Gapdh1 was analyzed as an internal control. (D) Relative mRNA levels of Wnt5a during EB formation from day0 to day10. Actin was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (E) Recombinant Wnt5 were supplemented to the Mst-/-EB culture from day 2 and day 10. Wild type EBs and Mst-/- EBs were grown in non-Wnt5a supplemented medium as controls. The percentage of beating EBs was profiled on day 8 and day 10 after initiating EBs culture. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).
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pone-0079867-g007: ES cell to cardiac progenitor cell differentiation is disturbed by Mst1/Mst2 depletion.(A) Heatmap of the expression of non-canonical Wnt signaling ligands (Wnt2, Wnt2b and Wnt5a) and canonical Wnt ligands (Wnt1, Wnt3a, Wnt8a and Wnt11) in day 4 and day 8 wild type EBs and Mst-/- EBs. (B) Relative mRNA levels of β-catenin in wild type and Mst-/- EBs at day 4 and day 8 during EB formation. Actin was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Immunoblotting analysis with antibodies against Active β-catenin and total β-catenin to check its expression in day 4 and day 8 wild type EBs and Mst-/- EBs. Gapdh1 was analyzed as an internal control. (D) Relative mRNA levels of Wnt5a during EB formation from day0 to day10. Actin was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (E) Recombinant Wnt5 were supplemented to the Mst-/-EB culture from day 2 and day 10. Wild type EBs and Mst-/- EBs were grown in non-Wnt5a supplemented medium as controls. The percentage of beating EBs was profiled on day 8 and day 10 after initiating EBs culture. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).
Mentions: To check whether there is any link between the defect of Mst-/- ES cell cardiac lineage differentiation and the Wnt signaling abnormal regulation, we examined the expression of Wnt ligand genes in day 4 and day 8 wild type and Mst-/- EBs. The expression of canonical Wnt ligand gene Wnt1 and Wnt3a showed no change between in Mst-/- EBs and wild type EBs, while another canonical Wnt ligand gene Wnt8a was slightly increased (Figure 7A). RT-PCR and western blot to examine their downstream mediator β-catenin revealed that total and active β-catenin were at the similar level in wild type EBs and Mst-/- EBs, indicating the defect of Mst-/- ES cell to cardiac lineage differentiation may not be related to canonical Wnt signaling (Figure 7B and 7C). On the other hand, several non-canonical Wnt ligand genes, such as Wnt2, Wnt2b, and Wnt5a, were significantly downregulated in Mst-/- EBs (Figure 7A). Exogenous Wnt2 can enhance ES cell to cardiomyocyte differentiation. To validate whether Wnt5a can also enhance the differentiation, we first examined Wnt5a expression during wild type EB differentiation. We found that Wnt5a was dramatically increased in day 4 EBs (Figure 7D). Therefore we added Wnt5a recombinant protein to day 2 Mst-/- EB culture. Notably, Mst-/- EBs grown in medium supplemented with Wnt5a showed increase number of beating EBs compared to Mst-/- EBs grown in non-Wnt5a medium on day 8 and day 10, although less than wild type EBs, indicating that Wnt5a can partially rescue Mst-/- EB defect for cardiomyocyte differentiation (Figure 6B and 7E). Taken together, these data suggest that Mst1/Mst2 may involve in cardiomyocyte differentiation through crosstalking with non-canonical Wnt signaling.

Bottom Line: The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation.They also proliferate faster than wild type ES cells.Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Pathology, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China.

ABSTRACT
The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived Mst1/Mst2 double knockout (Mst-/-) ES cells to completely perturb Hippo signaling. We found that Mst-/- ES cells express higher level of Nanog than wild type ES cells and show differentiation resistance after LIF withdrawal. They also proliferate faster than wild type ES cells. Although Mst-/- ES cells can form embryoid bodies (EBs), their differentiation into tissues of three germ layers is distorted. Intriguingly, Mst-/- ES cells are unable to form teratoma. Mst-/- ES cells can differentiate into mesoderm lineage, but further differentiation to cardiac lineage cells is significantly affected. Microarray analysis revealed that ligands of non-canonical Wnt signaling, which is critical for cardiac progenitor specification, are significantly repressed in Mst-/- EBs. Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation.

Show MeSH
Related in: MedlinePlus