Nakahara Masako, Nakamura Naoko, Matsuyama Satoko, Yogiashi Yoshiko, Yasuda Kazuki, Kondo Yasushi, Yuo Akira, Saeki Kumiko
Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan.
Cloning Stem Cells. 2009 Dec;11(4):509-22. doi: 10.1089/clo.2009.0023.
We previously reported a feeder-free culture method for pure production of subculturable vascular endothelial cells (VECs) from cynomolgus monkey embryonic stem cells (cmESCs) without as using cell-sorting technique. By this method, canonical vascular endothelial (VE)-cadherin/platelet-endothelial cell adhesion molecule 1 (PECAM1)-positive VECs (c-VECs) and atypical VE-cadherin/PECAM1-negative VECs (a-VECs) were generated without a contamination by pericytes, lymphatic endothelial cells, or immature ES cells. More recently, we established a unique culture technique to maintain human ESCs (hESCs) under a feeder-free and recombinant cytokine-free condition. Combining these two systems, we have successfully generated pure VECs from two lines of hESCs, khES-1 and khES-3, under a completely feeder-free condition. Our method is very simple: spheres generated from hESCs by floating culture using differentiation media supplemented with vascular endothelial growth factor, bone morphogenetic protein 4, stem cell factor, FMS-related tyrosine kinase-3 ligand, and interleukin 3 (IL3) and IL6 were cultured on gelatin-coated plates. Cell passage was performed by an ordinary enzymatic treatment. The hESC-derived differentiated cells demosntrated cord-forming activities and acetylated low-density lipoprotein-uptaking capacities. Moreover, they exclusively expressed von Willebrand factor and endothelial nitric oxide synthase. Flow cytometric analyses indicate that khES-3 generated both c-VECs and a-VECs as in the case of cmESCs. By contrast, khES-1 produced only a-VECs, which nonetheless demonstrated effective recruitment into neovascularity in vivo. Interestingly, a-VECs turned to express PECAM1 after transplantation into immunodeficient mice. The hESC-derived VECs were subculturable at least up to 10 passages without functional depression. Our method does not require a presorting processes to enrich progenitor fractions such as CD34-positive or kinase insert domain receptor (KDR)-positive cells, providing the most efficient and easiest technique for VEC production from hESCs.
我们之前报道了一种无需滋养层的培养方法，可从食蟹猴胚胎干细胞（cmESCs）中纯生产可传代培养的血管内皮细胞（VECs），且不使用细胞分选技术。通过这种方法，可生成典型的血管内皮（VE）-钙黏蛋白/血小板内皮细胞黏附分子1（PECAM1）阳性的VECs（c-VECs）和非典型的VE-钙黏蛋白/PECAM1阴性的VECs（a-VECs），且不受周细胞、淋巴管内皮细胞或未成熟ES细胞的污染。最近，我们建立了一种独特的培养技术，可在无滋养层和无重组细胞因子的条件下维持人胚胎干细胞（hESCs）。将这两个系统结合起来，我们已在完全无滋养层的条件下成功从两株hESCs，即khES-1和khES-3中生成了纯VECs。我们的方法非常简单：使用添加有血管内皮生长因子、骨形态发生蛋白4、干细胞因子、FMS相关酪氨酸激酶-3配体以及白细胞介素3（IL3）和IL6的分化培养基通过悬浮培养从hESCs生成的球体，在明胶包被的培养皿上进行培养。通过常规酶处理进行细胞传代。hESC来源的分化细胞表现出形成血管的活性和摄取乙酰化低密度脂蛋白的能力。此外，它们仅表达血管性血友病因子和内皮型一氧化氮合酶。流式细胞术分析表明，khES-3与cmESCs一样生成了c-VECs和a-VECs。相比之下，khES-1仅产生a-VECs，不过这些a-VECs在体内显示出有效募集到新生血管中的能力。有趣的是，a-VECs在移植到免疫缺陷小鼠后转变为表达PECAM1。hESC来源的VECs至少可传代培养10代而无功能衰退。我们的方法不需要预先分选过程来富集祖细胞组分，如CD34阳性或激酶插入结构域受体（KDR）阳性细胞，为从hESCs生产VECs提供了最有效且最简单的技术。
