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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2070.)
© 2006 American Heart Association, Inc.

Vascular Biology

Differentiation of Lymphatic Endothelial Cells From Embryonic Stem Cells on OP9 Stromal Cells

Tomoya Kono; Hajime Kubo; Chikashi Shimazu; Yoshihide Ueda; Meiko Takahashi; Kentoku Yanagi; Naoya Fujita; Takashi Tsuruo; Hiromi Wada; Jun K. Yamashita

From the Molecular and Cancer Research Unit, HMRO (T.K., H.K., Y.U., M.T.), and Department of Thoracic Surgery (T.K., H.W.), Graduate School of Medicine, Kyoto University, Japan; Laboratory of Stem Cell Differentiation (C.S., K.Y., J.K.Y.), Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Japan; Institute of Molecular and Cellular Biosciences (N.F., T.T.), The University of Tokyo, Japan; and PRESTO (J.K.Y.), Japan Science and Technology Agency, Japan.

Correspondence to Hajime Kubo, Molecular and Cancer Research Unit, HMRO, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan. E-mail kuboflt{at}kuhp.kyoto-u.ac.jp; or Jun K. Yamashita, Laboratory of Stem Cell Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Shogoin-Kawahara-cho53, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail juny@frontier.kyoto-u.ac.jp

Objectives— The discovery of vascular endothelial growth factor C (VEGF-C) and VEGF receptor-3 (VEGFR-3) has started to provide an understanding of the molecular mechanisms of lymphangiogenesis. The homeobox gene prox1 has been proven to specify lymphatic endothelial cells (ECs) from blood ECs. We investigated the process of lymphatic EC (LEC) differentiation using embryonic stem (ES) cells.

Methods and Results— VEGFR-2⁺ cells derived from ES cells differentiated into LECs at day 3 on OP9 stromal cells defined by the expression of prox1, VEGFR-3, and another lymphatic marker podoplanin. VEGFR-2⁺ cells gave rise to LYVE-1⁺ embryonic ECs, which were negative for prox1 on day 1 but turned to prox1⁺ LECs by day 3. VEGFR-3-Fc or Tie2-Fc, sequestering VEGF-C or angiopoietin1 (Ang1), suppressed colony formation of LECs on OP9 cells. However, addition of VEGF-C and Ang1 in combination with VEGF to the culture of VEGFR-2⁺ cells on collagen-coated dishes failed to induce LECs. LEC-inducing activity of OP9 cells was fully reproduced on paraformaldehyde-fixed OP9 cells with the conditioned medium.

Conclusion— We succeeded in differentiating LECs from ES cells and revealed the requirements of VEGF-C, Ang1, and other unknown factors for LEC differentiation.

To understand the process of the differentiation of lymphatic endothelial cells (LECs), we succeeded in differentiating LECs from embryonic stem (ES) cells, revealing the requirements of VEGF-C, Ang1, and other unknown factors for LEC differentiation. This ES cell system would be useful for better understanding the process of LEC differentiation.

Key Words: lymphatic endothelial cells • embryonic stem cells • prox1 • VEGF-C • VEGFR3

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