Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells
Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells
复制标题
DOI：
10.1002/jnr.20469
复制
发表时间：
2005-05-15
影响因子：
4.2
通讯作者：
Sakai, N
中科院分区：
文献类型：
--
作者：
Kitajima, H;Yoshimura, S;Sakai, N
胚胎干细胞在体外增殖并保持其多能性超过1年，因此可能为细胞治疗提供足够的来源。然而，以前报道的大多数获得细胞疗法来源的方法并不简单。在这里，我们描述了一种新的方法，通过在PA6细胞上共培养来从小鼠ES细胞诱导出神经球，而不是形成类胚体。ES细胞与PA6基质细胞共培养至少3d后，可分化为球状细胞。微球中的细胞均呈绿色荧光蛋白(GFP)阳性，表明它们来自表达绿色荧光蛋白的D3-ES细胞。球体内含有巢蛋白阳性细胞。PA6与PA6共培养时间越长，球体数量越多。即使经过10次机械分离和继代培养，仍能观察到球体的形成，显示出其自我更新能力。细胞分化为微管相关蛋白-2(MAP2)阳性的神经细胞和胶质纤维酸性蛋白(GFAP)阳性的胶质细胞。球体内还可见γ-氨基丁酸阳性细胞和酪氨酸羟化酶阳性细胞。PA6细胞共培养的时间不同，球体内MAP2或GFAP阳性细胞的百分比也不同。在共培养的早期阶段，会产生更多的神经元，在后期，会产生更多的神经胶质细胞。这些结果表明，胚胎干细胞可以通过与PA6共培养而生成神经球，这些细胞类似于来自小鼠胎脑组织的神经干细胞。(C)2005年Wiley-Liss，Inc.
Embryonic stem (ES) cells proliferate and maintain their pluripotency for over 1 year in vitro and may therefore provide a sufficient source for cell therapies. However, most of the previously reported methods for obtaining a source for cell therapies have not been simple. We describe here a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells cocultured with the PA6 stromal cell line for at least 3 days were capable of differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP-expressing D3-ES cells. The spheres contained nestin-positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self-renewal ability. The cells differentiated into microtubule-associated protein-2 (MAP2)-positive neuronal cells and glial fibrillary acidic protein (GFAP)-positive glial cells. gamma-Aminobutyric acid-positive cells and tyrosine hydroxylase-positive cells were also observed in the spheres. The percentages of the MAP2- or GFAP-positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue. (c) 2005 Wiley-Liss, Inc.