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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

Isolation of Mst-/- ES cells.(A) Genotyping of wild type (WT) ES cells and Mst-/- ES cells derived from blastocysts by PCR amplification of genomic DNA. Wild type ES cells showed a larger band while Mst-/- ES cells displayed a smaller band. Actin was used as an internal control. (B) Phase contrast microscopy of wild type (WT) and two independent Mst-/- knockout ES cell lines (Mst-/-1 and Mst-/-2) grown on 0.2% gelatin in 2i+LIF medium (Upper). These cells were stained for alkaline phosphatase (Lower). Scale bar, 200 μm. (C) mRNA level of Mst1 and Mst2 in wild type ES cells and Mst-/- ES cells examined by quantitative real-time PCR using primers flanking the deleted region of Mst1 and Mst2. The data are shown as the mean ± S.D (n=3). Actin was normalized as an internal control. Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunoblotting analysis of the expression of Mst1 and Mst2 in wild type ES cells and Mst-/- ES cells. Gapdh1 was used as a loading control.
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pone-0079867-g001: Isolation of Mst-/- ES cells.(A) Genotyping of wild type (WT) ES cells and Mst-/- ES cells derived from blastocysts by PCR amplification of genomic DNA. Wild type ES cells showed a larger band while Mst-/- ES cells displayed a smaller band. Actin was used as an internal control. (B) Phase contrast microscopy of wild type (WT) and two independent Mst-/- knockout ES cell lines (Mst-/-1 and Mst-/-2) grown on 0.2% gelatin in 2i+LIF medium (Upper). These cells were stained for alkaline phosphatase (Lower). Scale bar, 200 μm. (C) mRNA level of Mst1 and Mst2 in wild type ES cells and Mst-/- ES cells examined by quantitative real-time PCR using primers flanking the deleted region of Mst1 and Mst2. The data are shown as the mean ± S.D (n=3). Actin was normalized as an internal control. Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunoblotting analysis of the expression of Mst1 and Mst2 in wild type ES cells and Mst-/- ES cells. Gapdh1 was used as a loading control.

Mentions: Based on the schematics of alleles of Mst1 and Mst2 generated in a previous study [12], we crossed the Mst1+/-Mst2-/- male and female mice, harvested the E3.5 embryos and derived ES cells on MEF feeder (Figure S1A and S1B). These cells were further adapted to form feeder-free ES cell lines under the 2i+LIF condition for genotyping. Exon 4 and 5 which encode kinase domain are deleted in Mst1 knockout, while exon 5 and 6 which encode kinase domain are deleted in Mst2 knockout (Figure S1C). With primers targeted to the adjacent sequence of the deleted regions of Mst1 (exon 4 and 5) and Mst2 (exon 5 and 6), PCR confirmed that respective regions of Mst1 and Mst2 genomic DNA were deleted in Mst1/Mst2 double knockout (Mst-/-) ES cell lines respectively (Figure 1A). Two Mst1/Mst2 double knockout lines, Mst-/-1 and Mst-/-2 ES cell lines were selected for further studies (Figure 1A). The Mst-/- ES cells still maintained dome-shaped colony morphology similar to that of the wild type ES cells (Figure 1B). They also expressed high levels of ES protein alkaline phosphatase (Figure 1B). To further confirm the absence of both Mst1 and Mst2 in the Mst-/- ES cells, primers targeting the corresponding deleted transcript region were used to do RT-PCR, the result confirmed that neither Mst1 nor Mst2 transcript was found in Mst-/- ES cells (Figure 1C). Further examination of the protein extracts with antibodies specifically against kinase domains of Mst1 and Mst2 protein showed that neither Mst1 nor Mst2 proteins were detected in the Mst-/- ES cells (Figure 1D). Taken together, these results showed that both Mst1 and Mst2 were functionally inactive in both Mst-/- ES cell lines.


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)

Isolation of Mst-/- ES cells.(A) Genotyping of wild type (WT) ES cells and Mst-/- ES cells derived from blastocysts by PCR amplification of genomic DNA. Wild type ES cells showed a larger band while Mst-/- ES cells displayed a smaller band. Actin was used as an internal control. (B) Phase contrast microscopy of wild type (WT) and two independent Mst-/- knockout ES cell lines (Mst-/-1 and Mst-/-2) grown on 0.2% gelatin in 2i+LIF medium (Upper). These cells were stained for alkaline phosphatase (Lower). Scale bar, 200 μm. (C) mRNA level of Mst1 and Mst2 in wild type ES cells and Mst-/- ES cells examined by quantitative real-time PCR using primers flanking the deleted region of Mst1 and Mst2. The data are shown as the mean ± S.D (n=3). Actin was normalized as an internal control. Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunoblotting analysis of the expression of Mst1 and Mst2 in wild type ES cells and Mst-/- ES cells. Gapdh1 was used as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818222&req=5

pone-0079867-g001: Isolation of Mst-/- ES cells.(A) Genotyping of wild type (WT) ES cells and Mst-/- ES cells derived from blastocysts by PCR amplification of genomic DNA. Wild type ES cells showed a larger band while Mst-/- ES cells displayed a smaller band. Actin was used as an internal control. (B) Phase contrast microscopy of wild type (WT) and two independent Mst-/- knockout ES cell lines (Mst-/-1 and Mst-/-2) grown on 0.2% gelatin in 2i+LIF medium (Upper). These cells were stained for alkaline phosphatase (Lower). Scale bar, 200 μm. (C) mRNA level of Mst1 and Mst2 in wild type ES cells and Mst-/- ES cells examined by quantitative real-time PCR using primers flanking the deleted region of Mst1 and Mst2. The data are shown as the mean ± S.D (n=3). Actin was normalized as an internal control. Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunoblotting analysis of the expression of Mst1 and Mst2 in wild type ES cells and Mst-/- ES cells. Gapdh1 was used as a loading control.
Mentions: Based on the schematics of alleles of Mst1 and Mst2 generated in a previous study [12], we crossed the Mst1+/-Mst2-/- male and female mice, harvested the E3.5 embryos and derived ES cells on MEF feeder (Figure S1A and S1B). These cells were further adapted to form feeder-free ES cell lines under the 2i+LIF condition for genotyping. Exon 4 and 5 which encode kinase domain are deleted in Mst1 knockout, while exon 5 and 6 which encode kinase domain are deleted in Mst2 knockout (Figure S1C). With primers targeted to the adjacent sequence of the deleted regions of Mst1 (exon 4 and 5) and Mst2 (exon 5 and 6), PCR confirmed that respective regions of Mst1 and Mst2 genomic DNA were deleted in Mst1/Mst2 double knockout (Mst-/-) ES cell lines respectively (Figure 1A). Two Mst1/Mst2 double knockout lines, Mst-/-1 and Mst-/-2 ES cell lines were selected for further studies (Figure 1A). The Mst-/- ES cells still maintained dome-shaped colony morphology similar to that of the wild type ES cells (Figure 1B). They also expressed high levels of ES protein alkaline phosphatase (Figure 1B). To further confirm the absence of both Mst1 and Mst2 in the Mst-/- ES cells, primers targeting the corresponding deleted transcript region were used to do RT-PCR, the result confirmed that neither Mst1 nor Mst2 transcript was found in Mst-/- ES cells (Figure 1C). Further examination of the protein extracts with antibodies specifically against kinase domains of Mst1 and Mst2 protein showed that neither Mst1 nor Mst2 proteins were detected in the Mst-/- ES cells (Figure 1D). Taken together, these results showed that both Mst1 and Mst2 were functionally inactive in both Mst-/- ES cell lines.

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