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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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Cardiac Marker expression on Day 10 following treatment on days 4–6.qRT-PCR data showing expression of a panel of general and region-specific cardiac markers at day 10 after treatment of EBs with CM on days 4–6. (* indicates p<0.05).
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pone-0013446-g004: Cardiac Marker expression on Day 10 following treatment on days 4–6.qRT-PCR data showing expression of a panel of general and region-specific cardiac markers at day 10 after treatment of EBs with CM on days 4–6. (* indicates p<0.05).

Mentions: We also determined the expression of these markers at day 10 (Figure 4) and noted that only EBs treated with XEN-CM continued to show statistically significant increases in the expression of cardiac markers including, MHCα, Tbx5, cTnI, cTnT, Tbx5, Mlc2a and Mlc1a, Mlc2v. While the expression of regional-specific markers has not been rigorously assessed during EB differentiation, we noted that most of the markers upregulated show biased expression toward the atria or inflow tract of the embryonic heart (MHCα, Mlc1a, Mlc2a, cTnI [33], Tbx5 [34], [35]), whereas markers that are more strongly expressed in the ventricles (Hand1 [36], [37], Hand2 [37] and Irx4 [38]) were not significantly altered as compared to controls. Expression of the conduction system marker Connexin-40 (Cx40) [39], and the secondary heart field marker, Islet1 [40] (data not shown) were also not significantly different between treated EBs and controls. Taken together these data suggest that factors in XEN-CM may bias cardiomyocyte differentiation toward an atrial fate. To further address this, we analyzed the expression of atrial specific markers ANF/Nppa and Shox2, which mark the whole atrium and the right atrial wall, respectively. Nppa was not significantly different between treated and untreated EBs (not shown) and only PYS2-CM treated EBs showed a statistically significant increase in Shox2 expression, which marks the sinus venosus, and later, the dorsal wall of the right atrium [41]. Together, these findings suggest that endodermal CMs do not affect the overall patterning of the myocardium.


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)

Cardiac Marker expression on Day 10 following treatment on days 4–6.qRT-PCR data showing expression of a panel of general and region-specific cardiac markers at day 10 after treatment of EBs with CM on days 4–6. (* indicates p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013446-g004: Cardiac Marker expression on Day 10 following treatment on days 4–6.qRT-PCR data showing expression of a panel of general and region-specific cardiac markers at day 10 after treatment of EBs with CM on days 4–6. (* indicates p<0.05).
Mentions: We also determined the expression of these markers at day 10 (Figure 4) and noted that only EBs treated with XEN-CM continued to show statistically significant increases in the expression of cardiac markers including, MHCα, Tbx5, cTnI, cTnT, Tbx5, Mlc2a and Mlc1a, Mlc2v. While the expression of regional-specific markers has not been rigorously assessed during EB differentiation, we noted that most of the markers upregulated show biased expression toward the atria or inflow tract of the embryonic heart (MHCα, Mlc1a, Mlc2a, cTnI [33], Tbx5 [34], [35]), whereas markers that are more strongly expressed in the ventricles (Hand1 [36], [37], Hand2 [37] and Irx4 [38]) were not significantly altered as compared to controls. Expression of the conduction system marker Connexin-40 (Cx40) [39], and the secondary heart field marker, Islet1 [40] (data not shown) were also not significantly different between treated EBs and controls. Taken together these data suggest that factors in XEN-CM may bias cardiomyocyte differentiation toward an atrial fate. To further address this, we analyzed the expression of atrial specific markers ANF/Nppa and Shox2, which mark the whole atrium and the right atrial wall, respectively. Nppa was not significantly different between treated and untreated EBs (not shown) and only PYS2-CM treated EBs showed a statistically significant increase in Shox2 expression, which marks the sinus venosus, and later, the dorsal wall of the right atrium [41]. Together, these findings suggest that endodermal CMs do not affect the overall patterning of the myocardium.

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