Kono Tomoya, Kubo Hajime, Shimazu Chikashi, Ueda Yoshihide, Takahashi Meiko, Yanagi Kentoku, Fujita Naoya, Tsuruo Takashi, Wada Hiromi, Yamashita Jun K
Molecular and Cancer Research Unit, HMRO, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
Arterioscler Thromb Vasc Biol. 2006 Sep;26(9):2070-6. doi: 10.1161/01.ATV.0000225770.57219.b0. Epub 2006 May 11.
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.
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.
We succeeded in differentiating LECs from ES cells and revealed the requirements of VEGF-C, Ang1, and other unknown factors for LEC differentiation.
血管内皮生长因子C（VEGF-C）和VEGF受体-3（VEGFR-3）的发现开始让人们了解淋巴管生成的分子机制。同源框基因prox1已被证明可将血液内皮细胞（ECs）特化为淋巴管内皮细胞。我们利用胚胎干细胞（ES细胞）研究了淋巴管内皮细胞（LEC）的分化过程。
源自ES细胞的VEGFR-2+细胞在OP9基质细胞上培养3天时分化为LEC，其依据为prox1、VEGFR-3和另一种淋巴管标志物血小板内皮细胞黏附分子-1的表达情况。VEGFR-2+细胞产生LYVE-1+胚胎ECs，其在第1天时prox1呈阴性，但到第3天时转变为prox1+LEC。VEGFR-3-Fc或Tie2-Fc可隔离VEGF-C或血管生成素1（Ang1），从而抑制LEC在OP9细胞上的集落形成。然而，在胶原包被培养皿上培养VEGFR-2+细胞时，将VEGF-C和Ang1与VEGF联合添加并不能诱导产生LEC。OP9细胞的LEC诱导活性在经多聚甲醛固定的OP9细胞与条件培养基共同作用下可完全重现。
我们成功地从ES细胞中分化出LEC，并揭示了VEGF-C、Ang1和其他未知因子对LEC分化的需求。
