High-Efficiency Production of Subculturable Vascular Endothelial Cells from Feeder-Free Human Embryonic Stem Cells Without Cell-Sorting Technique
High-Efficiency Production of Subculturable Vascular Endothelial Cells from Feeder-Free Human Embryonic Stem Cells Without Cell-Sorting Technique
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DOI：
10.1089/clo.2009.0023
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发表时间：
2009-12-01
期刊：
影响因子：
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通讯作者：
Saeki, Kumiko
中科院分区：
文献类型：
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作者：
Nakahara, Masako;Nakamura, Naoko;Saeki, Kumiko
我们之前报道了一种无饲养层培养方法，用于从食蟹猴胚胎干细胞（cmESC）中纯生产可传代的血管内皮细胞（VEC），而不使用细胞分选技术。通过这种方法，产生了经典血管内皮（VE）-钙粘蛋白/血小板-内皮细胞粘附分子1（PECAM1）阳性VEC（c-VEC）和非典型VE-钙粘蛋白/PECAM1阴性VEC（a-VEC），而没有受到周细胞、淋巴内皮细胞或未成熟ES细胞的污染。最近，我们建立了一种独特的培养技术，可在无饲养层和重组无细胞因子的条件下维持人类ESC（hESC）。结合这两个系统，我们在完全无饲养层的条件下成功地从两个 hESC 系 khES-1 和 khES-3 中生成了纯 VEC。我们的方法非常简单：使用补充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的分化培养基通过漂浮培养从hESCs产生的球体在明胶包被的平板上培养。通过普通酶处理进行细胞传代。 hESC 衍生的分化细胞表现出索形成活性和乙酰化低密度脂蛋白摄取能力。此外，它们专门表达血管性血友病因子和内皮一氧化氮合酶。流式细胞术分析表明，khES-3 与 cmESC 一样产生 c-VEC 和 a-VEC。相比之下，khES-1 只产生 a-VEC，尽管如此，它仍能有效募集到体内新血管中。有趣的是，a-VEC 在移植到免疫缺陷小鼠体内后转而表达 PECAM1。 hESC 衍生的 VEC 可以传代至少 10 代，而不会出现功能性抑制。我们的方法不需要预分选过程来富集祖细胞组分，例如 CD34 阳性或激酶插入域受体 (KDR) 阳性细胞，为从 hESC 生产 VEC 提供了最有效和最简单的技术。
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.