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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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Effect of CM on cardiomyocyte differentiation when added before mesoderm formation.A, C. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes in control EBs. A separate control is included for END2 cells since they are grown in different medium from the other cell lines. B, D, E. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes after treatment on days 2–4 with (B) END2, (D) PYS2 and (E) XEN-CM. F. Summary of flow cytometry data showing the fold change in the number of MHCα::GFP (+) cells on days 10 and 13 after addition of CM on days 2–4. (*indicates a p<0.05). G, H. qRT-PCR data showing expression of mesoderm markers at days 4 and cardiac markers on day 7 after treatment of EBs with CM on days 2–4. (* indicates p<0.05).
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pone-0013446-g006: Effect of CM on cardiomyocyte differentiation when added before mesoderm formation.A, C. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes in control EBs. A separate control is included for END2 cells since they are grown in different medium from the other cell lines. B, D, E. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes after treatment on days 2–4 with (B) END2, (D) PYS2 and (E) XEN-CM. F. Summary of flow cytometry data showing the fold change in the number of MHCα::GFP (+) cells on days 10 and 13 after addition of CM on days 2–4. (*indicates a p<0.05). G, H. qRT-PCR data showing expression of mesoderm markers at days 4 and cardiac markers on day 7 after treatment of EBs with CM on days 2–4. (* indicates p<0.05).

Mentions: Studies of cardiac specification in various animal models reveal a strict temporal requirement for signals from the endoderm involved in this process [1], [42]. To test this, EBs were treated with CM in an earlier time window, between days 2–4, which corresponds to a period just prior to mesoderm formation in our cultures (Figure 5A, B). One possible mechanism by which endodermal signals might enhance myocardial differentiation is by increasing mesoderm formation. If this were the case then it might be expected that addition of CMs at this earlier time point might further increase cardiac differentiation. As before, we tested whether addition of CM could increase the percentage of EBs that formed beating areas. EBs were assessed from day 6 (first day of beating) until day 21, for beating and activity of the MHCα::GFP reporter. Approximately 50 EBs were scored each day, and beating was represented as a percentage of all EBs scored (Figure 5C: line graphs) to show the overall trend in beating within the cultures. Beating was also normalized to controls and represented on each day as a percentage of beating in controls on that day (Figure 5D: bar graphs). In contrast to the results obtained when CM was added during the peak of Brachyury expression, when EBs were treated from day 2–4 (prior to mesoderm formation) CM either had no effect on, or delayed the onset of beating (Figure 5D). Only XEN-CM enhanced beating when added in this time window. To confirm this, we performed flow cytometry on cells isolated from day 10 EBs using MHCα::GFP reporter for cells possessing a cardiac fate (Figure 6A–E). GFP-positive cells were analyzed and assessed as a percentage of total cells counted. Addition of endodermal-CM from days 2–4 had either no impact or a negative impact on the percentage of GFP-positive cells at day 10 as compared to controls (Figure 6F). This was consistent with EB counting data on day 10. These cultures were also analyzed by flow cytometry at day 13. There was a small but statistically significant increase after treatment with PYS2-CM, but otherwise there were no differences between treated and untreated EBs at these time points (Figure 6F). As before, we also assessed myocardial specification in response to endodermal CM by using qRT-PCR to assess for changes in the expression of mesoderm markers generally and cardiac markers specifically. At day 4, (just after the termination of CM treatment) we examined the expression of markers for the nascent mesoderm (Figure 6G). None of the CMs increased expression of Brachyury, but addition of XEN-CM in this time window did result in a statistically significant increase of Fgf8 expression.


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)

Effect of CM on cardiomyocyte differentiation when added before mesoderm formation.A, C. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes in control EBs. A separate control is included for END2 cells since they are grown in different medium from the other cell lines. B, D, E. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes after treatment on days 2–4 with (B) END2, (D) PYS2 and (E) XEN-CM. F. Summary of flow cytometry data showing the fold change in the number of MHCα::GFP (+) cells on days 10 and 13 after addition of CM on days 2–4. (*indicates a p<0.05). G, H. qRT-PCR data showing expression of mesoderm markers at days 4 and cardiac markers on day 7 after treatment of EBs with CM on days 2–4. (* indicates p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958120&req=5

pone-0013446-g006: Effect of CM on cardiomyocyte differentiation when added before mesoderm formation.A, C. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes in control EBs. A separate control is included for END2 cells since they are grown in different medium from the other cell lines. B, D, E. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes after treatment on days 2–4 with (B) END2, (D) PYS2 and (E) XEN-CM. F. Summary of flow cytometry data showing the fold change in the number of MHCα::GFP (+) cells on days 10 and 13 after addition of CM on days 2–4. (*indicates a p<0.05). G, H. qRT-PCR data showing expression of mesoderm markers at days 4 and cardiac markers on day 7 after treatment of EBs with CM on days 2–4. (* indicates p<0.05).
Mentions: Studies of cardiac specification in various animal models reveal a strict temporal requirement for signals from the endoderm involved in this process [1], [42]. To test this, EBs were treated with CM in an earlier time window, between days 2–4, which corresponds to a period just prior to mesoderm formation in our cultures (Figure 5A, B). One possible mechanism by which endodermal signals might enhance myocardial differentiation is by increasing mesoderm formation. If this were the case then it might be expected that addition of CMs at this earlier time point might further increase cardiac differentiation. As before, we tested whether addition of CM could increase the percentage of EBs that formed beating areas. EBs were assessed from day 6 (first day of beating) until day 21, for beating and activity of the MHCα::GFP reporter. Approximately 50 EBs were scored each day, and beating was represented as a percentage of all EBs scored (Figure 5C: line graphs) to show the overall trend in beating within the cultures. Beating was also normalized to controls and represented on each day as a percentage of beating in controls on that day (Figure 5D: bar graphs). In contrast to the results obtained when CM was added during the peak of Brachyury expression, when EBs were treated from day 2–4 (prior to mesoderm formation) CM either had no effect on, or delayed the onset of beating (Figure 5D). Only XEN-CM enhanced beating when added in this time window. To confirm this, we performed flow cytometry on cells isolated from day 10 EBs using MHCα::GFP reporter for cells possessing a cardiac fate (Figure 6A–E). GFP-positive cells were analyzed and assessed as a percentage of total cells counted. Addition of endodermal-CM from days 2–4 had either no impact or a negative impact on the percentage of GFP-positive cells at day 10 as compared to controls (Figure 6F). This was consistent with EB counting data on day 10. These cultures were also analyzed by flow cytometry at day 13. There was a small but statistically significant increase after treatment with PYS2-CM, but otherwise there were no differences between treated and untreated EBs at these time points (Figure 6F). As before, we also assessed myocardial specification in response to endodermal CM by using qRT-PCR to assess for changes in the expression of mesoderm markers generally and cardiac markers specifically. At day 4, (just after the termination of CM treatment) we examined the expression of markers for the nascent mesoderm (Figure 6G). None of the CMs increased expression of Brachyury, but addition of XEN-CM in this time window did result in a statistically significant increase of Fgf8 expression.

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