Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors

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Abstract

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance1,2,3,4. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells5, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall6,7,8. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as ‘vascular progenitor cells’ to form mature vessels and thus offer potential for tissue engineering of the vascular system.

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Figure 1: Differentiation of Flk1+ cells into endothelial and mural cells.
Figure 2: Flk1+ cell differentiation in serum-free culture.
Figure 3: Single-cell deposition of Flk1+ cells cultivated with 10% FCS and 50 ng ml-1 VEGF for four days and stained for PECAM1 (purple) and SMA (1A4; brown).
Figure 4: Vascular formation of Flk1+ cell aggregates in three-dimensional culture with 10% serum and 50 ng ml-1 VEGF.
Figure 5: Contribution of Flk1+ cells to vascular formation in vivo.

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Acknowledgements

We thank M. J. Evans for CCE ES cells, N. Matsuyoshi for the hybridoma, A. Nagafuchi for antibodies, T. Kunisada for LacZ construct, R. Yu for critical reading of the manuscript, and many of our colleagues for suggestions and discussion. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, the Ministry of Health and Welfare of Japan (S.I.N.), Japanese Society for the Promotion of Science “Research for the Future” Program (H.I., M.O. and S.I.N.), Japan Tobacco Foundation, Japan Hearth Foundation & Pfizer Pharmaceuticals Grant for Research on Coronary Artery Disease and Tanabe Medical Frontier Conference. J.Y. is a recipient of the Research Fellowship grant of the Japan Society for the Promotion of Science for Young Scientists.

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Correspondence to Jun Yamashita.

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