A simple method for differentiation of H9 cells into neuroectoderm
A simple method for differentiation of H9 cells into neuroectoderm
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H9细胞分化为神经外胚层的简单方法
DOI：
10.1016/j.tice.2015.07.006
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发表时间：
2015-10-01
期刊：
影响因子：
2.6
通讯作者：
Liu, Chao
中科院分区：
文献类型：
--
作者：
Liu, Annuo;Zhang, Dijuan;Liu, Chao
人胚胎干细胞（ESC）可以形成神经外胚层（NE），为体外解剖NE形成提供平台。然而，人类 ESC 可以分化成所有三个胚层。因此，开发将人类ESC分化为NE细胞的有效方法至关重要。铺板细胞密度和局部细胞密度 (LCD) 都会影响 NE 分化。在这里，我们开发了一种基于细胞簇的 NE 分化方法，其中电镀细胞密度和 LCD 都受到控制。使用我们的新方法，高电镀细胞密度可促进 NE 标记蛋白 PAX6 的表达。两种 SMAD 信号阻断剂 SB431542 和 NOGGIN 下调 OCT4 并上调 PAX6，但在分化 5 天后不影响 GATA2 的 mRNA 表达。此外，IB 分析显示，在神经分化过程中，PAX6 和 β-III-微管蛋白呈时间依赖性上调，同时 OCT4 下调。还检测到 H9 衍生细胞中 TH 和 β-III-微管蛋白的共表达，证明 NE 细胞具有分化为特定神经元之一的能力。我们共同建立了一种从 H9 细胞生成 NE 细胞的简单方法，这可能有助于开发高效的神经分化方法。 (C) 2015 Elsevier Ltd. 保留所有权利。
Human embryonic stem cells (ESCs) can form neuroectoderm (NE), providing a platform for in vitro dissection of NE formation. However, human ESCs can differentiate into all three germ layers. It thus is crucial to develop efficient methods for differentiation of human ESCs into NE cells. Both plating cell density and localized cell density (LCD) affect NE differentiation. Here, we developed a cell cluster-based NE differentiation method, in which both plating cell density and LCD are under control. Using our new method, high plating cell densities promote expression of PAX6, a NE marker protein. Two SMAD signaling blockers, SB431542 and NOGGIN, downregulate OCT4 and upregulate PAX6, while does not affect mRNA expression of GATA2 after 5 d of differentiation. Moreover, IB analysis showed a time-dependent upregulation of PAX6 and beta-III-tubulin together with a downregulation of OCT4 during the neural differentiation. Coexpression of both TH and beta-III-tubulin in the H9-derived cells was also detected, proving the NE cells have an ability to differentiate into one of the specific neurons. Together, we established a simple method for generating NE cells from H9 cells, which might contribute to develop high efficient method for neural differentiation. (C) 2015 Elsevier Ltd. All rights reserved.