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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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Culture conditions that delay VE formation also block cardiac differentiation, but cardiac differentiation is partially rescued by the addition of endodermally conditioned medium.A. Pseudo-colored fluorescence images showing Afp::GFP expression (Green) during EB formation under normal serum-containing conditions (STD) and during serum-free conditions (SRM). Note that VE formation is delayed by approximately 2 days. B. Summary of Flow Cytometry data for MHCα::GFP at day 10 comparing EBs grown under standard condition to those grown under serum free conditions. Addition of all three endodermally conditioned media, but not MEF conditioned medium, had a positive impact on cardiomyocyte formation.
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pone-0013446-g012: Culture conditions that delay VE formation also block cardiac differentiation, but cardiac differentiation is partially rescued by the addition of endodermally conditioned medium.A. Pseudo-colored fluorescence images showing Afp::GFP expression (Green) during EB formation under normal serum-containing conditions (STD) and during serum-free conditions (SRM). Note that VE formation is delayed by approximately 2 days. B. Summary of Flow Cytometry data for MHCα::GFP at day 10 comparing EBs grown under standard condition to those grown under serum free conditions. Addition of all three endodermally conditioned media, but not MEF conditioned medium, had a positive impact on cardiomyocyte formation.

Mentions: To address the possibility that VE formation within the EB may mask the requirement for contact-dependent signals in cardiomyocyte formation, we empirically determined conditions in which VE formation within EBs was delayed (Figure 12A). Briefly, EB size was decreased and serum was replaced with a defined serum replacement. Under these conditions, we found that VE formation began approximately 2 days later than in controls. In this condition we also found that mesoderm formed robustly (as assessed by the expression of Brachyury), but that cardiac differentiation was severely impaired with only a small background of cells expressing the MHCα::GFP reporter and beating (data not shown). To test whether factors in the CM could rescue cardiac differentiation, we added CMs (also produced in serum-free media) at days 4–6 of EB differentiation (Figure 12B). At day 10 we analyzed the percentage of cells within the culture that underwent myocardial differentiation and noted a 2–3 fold increase in cardiomyocyte differentiation when EBs were treated with CM as compared to EBs grown in the serum-free media alone. However, the total number of myocardial cells that formed remained lower than those that formed under standard, serum containing, media conditions. This suggests that there are either contact-dependent signals produced by the VE or that there are factors in serum, but absent in serum-free media that are required for expansion of cardiac progenitors that form within EBs.


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)

Culture conditions that delay VE formation also block cardiac differentiation, but cardiac differentiation is partially rescued by the addition of endodermally conditioned medium.A. Pseudo-colored fluorescence images showing Afp::GFP expression (Green) during EB formation under normal serum-containing conditions (STD) and during serum-free conditions (SRM). Note that VE formation is delayed by approximately 2 days. B. Summary of Flow Cytometry data for MHCα::GFP at day 10 comparing EBs grown under standard condition to those grown under serum free conditions. Addition of all three endodermally conditioned media, but not MEF conditioned medium, had a positive impact on cardiomyocyte formation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958120&req=5

pone-0013446-g012: Culture conditions that delay VE formation also block cardiac differentiation, but cardiac differentiation is partially rescued by the addition of endodermally conditioned medium.A. Pseudo-colored fluorescence images showing Afp::GFP expression (Green) during EB formation under normal serum-containing conditions (STD) and during serum-free conditions (SRM). Note that VE formation is delayed by approximately 2 days. B. Summary of Flow Cytometry data for MHCα::GFP at day 10 comparing EBs grown under standard condition to those grown under serum free conditions. Addition of all three endodermally conditioned media, but not MEF conditioned medium, had a positive impact on cardiomyocyte formation.
Mentions: To address the possibility that VE formation within the EB may mask the requirement for contact-dependent signals in cardiomyocyte formation, we empirically determined conditions in which VE formation within EBs was delayed (Figure 12A). Briefly, EB size was decreased and serum was replaced with a defined serum replacement. Under these conditions, we found that VE formation began approximately 2 days later than in controls. In this condition we also found that mesoderm formed robustly (as assessed by the expression of Brachyury), but that cardiac differentiation was severely impaired with only a small background of cells expressing the MHCα::GFP reporter and beating (data not shown). To test whether factors in the CM could rescue cardiac differentiation, we added CMs (also produced in serum-free media) at days 4–6 of EB differentiation (Figure 12B). At day 10 we analyzed the percentage of cells within the culture that underwent myocardial differentiation and noted a 2–3 fold increase in cardiomyocyte differentiation when EBs were treated with CM as compared to EBs grown in the serum-free media alone. However, the total number of myocardial cells that formed remained lower than those that formed under standard, serum containing, media conditions. This suggests that there are either contact-dependent signals produced by the VE or that there are factors in serum, but absent in serum-free media that are required for expansion of cardiac progenitors that form within EBs.

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