Sone Masakatsu, Itoh Hiroshi, Yamashita Jun, Yurugi-Kobayashi Takami, Suzuki Yutaka, Kondo Yasushi, Nonoguchi Akane, Sawada Naoki, Yamahara Kenichi, Miyashita Kazutoshi, Park Kwijun, Shibuya Masabumi, Nito Shinji, Nishikawa Shin-Ichi, Nakao Kazuwa
Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507 Japan.
Circulation. 2003 Apr 29;107(16):2085-8. doi: 10.1161/01.CIR.0000070022.78747.1B. Epub 2003 Apr 21.
We demonstrated that vascular endothelial growth factor receptor 2 (VEGF-R2)-positive cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells and mural cells to suffice as vascular progenitor cells (VPCs). Here we examined whether VPCs occur in primate ES cells and investigated the differences in VPC differentiation kinetics between primate and mouse ES cells.
In contrast to mouse ES cells, undifferentiated monkey ES cells expressed VEGF-R2. By culturing these undifferentiated ES cells for 4 days on OP9 feeder layer, VEGF-R2 expression disappeared, and then reappeared after 8 days of differentiation. We then isolated these VEGF-R2-positive and vascular endothelial cadherin (VEcadherin)-negative cells by flow cytometry sorting. Additional 5-day reculture of these VEGF-R2+ VEcadherin- cells on OP9 feeder layer resulted in the appearance of platelet endothelial cell adhesion molecule-1 (PECAM1)-positive, VEcadherin-positive, endothelial nitric oxide synthase (eNOS)-positive endothelial cells. On a collagen IV-coated dish in the presence of serum, these cells differentiated into smooth muscle actin (SMA)-positive and calponin-positive mural cells (pericytes or vascular smooth muscle cells). Addition of 50 ng/mL VEGF to the culture on a collagen IV-coated dish resulted in the appearance of PECAM1+ cells surrounded by SMA+ cells. In addition, these differentiated VEGF-R2+ cells can form tube-like structures in a 3-dimensional culture.
Our findings indicate that differentiation kinetics of VPCs derived from primate and mouse ES cells were different. Differentiated VEGF-R2+ VEcadherin- cells can act as VPCs in primates. To seek the clinical potential of VPCs for vascular regeneration, investigations of primate ES cells are indispensable.
我们已证明，源自小鼠胚胎干细胞（ES细胞）的血管内皮生长因子受体2（VEGF-R2）阳性细胞可分化为内皮细胞和平滑肌细胞，足以作为血管祖细胞（VPCs）。在此，我们研究了灵长类ES细胞中是否存在VPCs，并探讨了灵长类和小鼠ES细胞在VPC分化动力学上的差异。
与小鼠ES细胞不同，未分化的猴ES细胞表达VEGF-R2。通过在OP9饲养层上培养这些未分化的ES细胞4天，VEGF-R2表达消失，然后在分化8天后重新出现。然后，我们通过流式细胞术分选分离出这些VEGF-R2阳性且血管内皮钙黏蛋白（VE钙黏蛋白）阴性的细胞。将这些VEGF-R2 + VE钙黏蛋白阴性细胞在OP9饲养层上再培养5天，导致血小板内皮细胞黏附分子-1（PECAM1）阳性、VE钙黏蛋白阳性、内皮型一氧化氮合酶（eNOS）阳性的内皮细胞出现。在存在血清的情况下，将这些细胞接种于胶原IV包被的培养皿上，它们分化为平滑肌肌动蛋白（SMA）阳性和钙调蛋白阳性的壁细胞（周细胞或血管平滑肌细胞）。在胶原IV包被的培养皿上的培养物中添加50 ng/mL VEGF，导致出现被SMA +细胞包围的PECAM1 +细胞。此外，这些分化的VEGF-R2 +细胞可在三维培养中形成管状结构。
我们的研究结果表明，源自灵长类和小鼠ES细胞的VPCs的分化动力学不同。分化的VEGF-R2 + VE钙黏蛋白阴性细胞可作为灵长类动物的VPCs。为探索VPCs在血管再生方面的临床潜力，对灵长类ES细胞的研究必不可少。
