Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium
Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium
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DOI：
10.1387/ijdb.130173to
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发表时间：
2013-01-01
影响因子：
0.7
通讯作者：
Okamoto, Tetsuji
中科院分区：
文献类型：
--
作者：
Yamasaki, Sachiko;Nabeshima, Kou;Okamoto, Tetsuji
小鼠胚胎干 (mES) 细胞和小鼠诱导多能干 (miPS) 细胞通常保存在灭活的小鼠胚胎成纤维细胞饲养细胞上，培养基中补充有胎牛血清或专有替代品。由于相对缺乏关于细胞对分化生长因子的反应的知识，含有未知量试剂的未定义培养基限制了多能细胞的应用开发。因此，我们开发了一种无血清培养基，命名为ESF7，其中mES细胞可以在没有饲养细胞的情况下维持在未分化状态。测试该培养基用于培养 miPS 细胞。 miPS细胞在ESF7培养基中保存3年以上，具有未分化表型，表现为多能性标记基因和碱性磷酸酶的表达；这些细胞表现出基本正常的核型。此外，我们发现成纤维细胞生长因子-2 (FGF-2) 与肝素一起诱导 miPS 细胞分化为神经元细胞，无论是在贴壁单层还是在胚状体悬浮培养中。此外，我们发现FGF-2与骨形态发生蛋白2在胚状体悬浮培养中诱导miPS细胞分化为心肌细胞。此外，我们将保存在ESF7中的miPS细胞皮下移植到SCID小鼠的背侧；所有移植都产生了肿瘤，其组织来自所有三个胚胎胚层。由于这种简单的无血清贴壁单一培养系统支持多能 iPS 细胞在体外的长期增殖，它将使我们能够阐明在特定条件下细胞对生长因子的反应，并且它应该为人类 iPS 细胞的分化方案提供有用的信息。
Mouse embryonic stem (mES) cells and mouse induced pluripotent stem (miPS) cells are commonly maintained on inactivated mouse embryonic fibroblast feeder cells in medium supplemented with fetal bovine serum or proprietary replacements. An undefined medium containing unknown quantities of reagents has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. Therefore we developed a serum-free medium, designated ESF7, in which mES cells can be maintained in an undifferentiated state without feeder cells. The medium was tested for culturing miPS cells. The miPS cells have been maintained in ESF7 medium for more than 3 years with an undifferentiated phenotype manifested by the expression of pluripotency marker genes and alkaline phosphatase; and these cells exhibited largely normal karyotypes. Furthermore, we found that fibroblast growth factor-2 (FGF-2) with heparin induced miPS cell differentiation into neuronal cells, both in an adherent monolayer and in embryoid body suspension culture. Moreover, we found that FGF-2 with bone morphogenetic protein 2 induced miPS cell differentiation into cardiomyocytes in embryoid body suspension culture. Furthermore, we transplanted subcutaneously miPS cells maintained in ESF7 into the dorsal flanks of SCID mice; all of the transplants produced tumors with tissues derived from all three embryonic germ layers. As this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent iPS cells in vitro, it will allow us to elucidate cell responses to growth factors under defined conditions, and it should provide useful information for differentiation protocols for human iPS cells.