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eXtraembryonic ENdoderm (XEN) stem cells produce factors that activate heart formation.

Brown K, Doss MX, Legros S, Artus J, Hadjantonakis AK, Foley AC - PLoS ONE (2010)

Bottom Line: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation.Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts.These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

View Article: PubMed Central - PubMed

Affiliation: Greenberg Division of Cardiology, Weill Cornell Medical College, New York, New York, United States of America.

ABSTRACT

Background: Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.

Methodology/principal findings: Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.

Conclusions/significance: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

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Immunocytochemistry of MHCα::GFP(+) cells induced by XEN-CM.EBs treated from days 4–6 with XEN-CM were dissociated as described for flow cytometry analysis. They were then fixed and processed for immunoctyochemistry using antibodies that recognize either GFP (green in A and B) or the cardiac specific epitopes Troponin (red in A and C), Cardiac Actin (red in B and D) and cardiac-specific Myosin (MF20) (green in C and D). These studies demonstrate the correlation between cardiac markers and GFP.
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pone-0013446-g003: Immunocytochemistry of MHCα::GFP(+) cells induced by XEN-CM.EBs treated from days 4–6 with XEN-CM were dissociated as described for flow cytometry analysis. They were then fixed and processed for immunoctyochemistry using antibodies that recognize either GFP (green in A and B) or the cardiac specific epitopes Troponin (red in A and C), Cardiac Actin (red in B and D) and cardiac-specific Myosin (MF20) (green in C and D). These studies demonstrate the correlation between cardiac markers and GFP.

Mentions: Our flow cytometry analysis of these cells was based on the expression of GFP driven by the MHCα promoter. This cell line has been previously described [30]; however, we wanted to confirm that the GFP reporter faithfully reflected cardiomyocyte differentiation and wanted to show that treatment with XEN-CM neither activates a non-cardiac cell type expressing the MHCα::GFP reporter nor increases the stability of GFP in cells that may have previously expressed cardiac markers transiently, but no longer do so. To address these possibilities, EBs that had been treated with XEN-CM were dissociated using the same protocol as was used for the flow cytometry studies, and plated onto gelatin-coated chamber slides. 24 hours after plating, cells were observed by fluorescence microscopy. At this point, GFP–positive cells were seen as rhythmically contracting cells in these dissociated cultures. By contrast, no contractions were observed in GFP-negative cells. Cells were then fixed and processed by immunocytochemistry for antibodies recognizing GFP and the cardiac-specific epitopes, Troponin, Cardiac Actin and Cardiac-specific Myosin (MF20). We noted that GFP-positive cells also expressed Troponin and Cardiac Actin (Figure 3A, B). There was also coexpression between these cardiac markers and cells recognized by the MF20 antibody, which recognizes cardiac-specific myosin (Figure 3C, D). These findings demonstrate that MHCα::GFP is a faithful reporter of cardiac fates and the addition of XEN-CM does not activate non-cardiac GFP or enhance the perdurance of GFP.


eXtraembryonic ENdoderm (XEN) stem cells produce factors that activate heart formation.

Brown K, Doss MX, Legros S, Artus J, Hadjantonakis AK, Foley AC - PLoS ONE (2010)

Immunocytochemistry of MHCα::GFP(+) cells induced by XEN-CM.EBs treated from days 4–6 with XEN-CM were dissociated as described for flow cytometry analysis. They were then fixed and processed for immunoctyochemistry using antibodies that recognize either GFP (green in A and B) or the cardiac specific epitopes Troponin (red in A and C), Cardiac Actin (red in B and D) and cardiac-specific Myosin (MF20) (green in C and D). These studies demonstrate the correlation between cardiac markers and GFP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958120&req=5

pone-0013446-g003: Immunocytochemistry of MHCα::GFP(+) cells induced by XEN-CM.EBs treated from days 4–6 with XEN-CM were dissociated as described for flow cytometry analysis. They were then fixed and processed for immunoctyochemistry using antibodies that recognize either GFP (green in A and B) or the cardiac specific epitopes Troponin (red in A and C), Cardiac Actin (red in B and D) and cardiac-specific Myosin (MF20) (green in C and D). These studies demonstrate the correlation between cardiac markers and GFP.
Mentions: Our flow cytometry analysis of these cells was based on the expression of GFP driven by the MHCα promoter. This cell line has been previously described [30]; however, we wanted to confirm that the GFP reporter faithfully reflected cardiomyocyte differentiation and wanted to show that treatment with XEN-CM neither activates a non-cardiac cell type expressing the MHCα::GFP reporter nor increases the stability of GFP in cells that may have previously expressed cardiac markers transiently, but no longer do so. To address these possibilities, EBs that had been treated with XEN-CM were dissociated using the same protocol as was used for the flow cytometry studies, and plated onto gelatin-coated chamber slides. 24 hours after plating, cells were observed by fluorescence microscopy. At this point, GFP–positive cells were seen as rhythmically contracting cells in these dissociated cultures. By contrast, no contractions were observed in GFP-negative cells. Cells were then fixed and processed by immunocytochemistry for antibodies recognizing GFP and the cardiac-specific epitopes, Troponin, Cardiac Actin and Cardiac-specific Myosin (MF20). We noted that GFP-positive cells also expressed Troponin and Cardiac Actin (Figure 3A, B). There was also coexpression between these cardiac markers and cells recognized by the MF20 antibody, which recognizes cardiac-specific myosin (Figure 3C, D). These findings demonstrate that MHCα::GFP is a faithful reporter of cardiac fates and the addition of XEN-CM does not activate non-cardiac GFP or enhance the perdurance of GFP.

Bottom Line: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation.Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts.These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

View Article: PubMed Central - PubMed

Affiliation: Greenberg Division of Cardiology, Weill Cornell Medical College, New York, New York, United States of America.

ABSTRACT

Background: Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.

Methodology/principal findings: Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.

Conclusions/significance: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

Show MeSH