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Notch signaling respecifies the hemangioblast to a cardiac fate

Abstract

To efficiently generate cardiomyocytes from embryonic stem (ES) cells in culture it is essential to identify key regulators of the cardiac lineage and to develop methods to control them. Using a tet-inducible mouse ES cell line to enforce expression of a constitutively activated form of the Notch 4 receptor, we show that signaling through the Notch pathway can efficiently respecify hemangioblasts to a cardiac fate, resulting in the generation of populations consisting of >60% cardiomyocytes. Microarray analyses reveal that this respecification is mediated in part through the coordinated regulation of the BMP and Wnt pathways by Notch signaling. Together, these findings have uncovered a potential role for the Notch pathway in cardiac development and provide an approach for generating large numbers of cardiac progenitors from ES cells.

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Figure 1: The role of Notch signaling in cardiac differentiation from embryoid body–derived Bry-GFP+/Flk-1 cardiac mesodermal cells.
Figure 2: Effects of Notch4 overexpression on the embryoid body–derived Bry-GFP+/Flk-1+ population.
Figure 3: Respecification by Notch signaling is restricted to the hemangioblast stage of embryoid body development.
Figure 4: Effect of Notch4 expression on BL-CFC–derived blast colony development.
Figure 5: Microarray-based expression analysis of Bry-GFP+/Flk-1+ cells after Dox induction.
Figure 6: Effects of cytokines on the cardiac differentiation from Bry-GFP+/Flk-1+ and Bry-GFP+/Flk-1 cells.
Figure 7

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Acknowledgements

We would like to thank members of the Keller laboratory for discussions and critical reading of the manuscript, Stefan Irion (McEwen Center for Regenerative Medicine) for providing the Bry-GFP/Ainv ES cell line, Kitajewski for providing the activated form of Notch4 cDNA (int-3)27 tagged with hemagglutinin (HA) sequence, M. Kyba (Lillehei Heart Institute, University of Minnesota) for the tet-on inducible ES cell line, Ainv18 and Zuniga-Pflucker (University of Toronto, Sunnybrook Research Institute) for the OP9-DL1 cell line. This work was supported by National Institutes of Health grants R01 HL71800, R01 HL 48834.

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V.C.C. and G.K. conceived the experiments and V.C.C. designed experimental details. R.S. and D.J. performed microarray analyses. X.C. generated the Notch1-inducible ES cell line and participated in experimental design. V.C.C. performed all remaining experiments. The manuscript was written by V.C.C. and G.K.

Corresponding author

Correspondence to Gordon Keller.

Supplementary information

Supplementary Text and Figures

Figures 1–4 (PDF 173 kb)

Supplementary Video 1

Day 3.25 Bry-GFP+/Flk-1+ cell reaggregated 24 hours in the presence of Dox and plated in the cardiac cultures (Original magnification 40x; QuickTime movie; 2.0 MB). (MOV 1991 kb)

Supplementary Video 2

Day 3.25 Bry-GFP+/Flk-1+ cells reaggregated in the absence of Dox and plated in the cardiac cultures (Original magnification 40x; QuickTime movie; 2.0 MB) (MOV 2049 kb)

Supplementary Video 3

Compact colony with contracting cells generated in the blast colony assay in the presence of Dox (Original magnification 200x; QuickTime movie; 0.13 MB) (MOV 133 kb)

Supplementary Video 4

Mixed lineage colony with contracting core surrounded by red outer cells (Original magnification 200x; QuickTime movie; 0.19 MB) (MOV 189 kb)

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Chen, V., Stull, R., Joo, D. et al. Notch signaling respecifies the hemangioblast to a cardiac fate. Nat Biotechnol 26, 1169–1178 (2008). https://doi.org/10.1038/nbt.1497

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