Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors
Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors
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DOI：
10.1016/j.bbrc.2017.06.039
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发表时间：
2017-08-19
影响因子：
3.1
通讯作者：
Ko, Minoru S. H.
中科院分区：
文献类型：
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作者：
Matsushita, Misako;Nakatake, Yuhki;Ko, Minoru S. H.
多能人胚胎干细胞（hESC）可以分化成多种细胞谱系，因此为研究细胞分化过程中的分子机制提供了最佳平台之一。最近，我们报道了通过引入编码五种转录因子（TF）的合成 mRNA 混合物，hESC 可以快速有效地分化为功能性神经元：NEUROGI、NEUROG2、NEUROG3、NEURODI 和 NEUROD2。在这里，我们进一步测试了一种可能性，即即使是单个转录因子，当异位表达时，也可以将 hESC 分化为神经元。为此，我们建立了 hESC 系，其中每个 TF 都可以通过多西环素诱导的 PiggyBac 载体过表达。这五个 TF 中任何一个的过度表达确实引起了 hESC 的快速且相当均匀的分化，根据其形态、qRT-PCR 和免疫组织化学，hESC 被鉴定为神经元。此外，钙成像分析和膜片钳记录表明这些分化细胞具有电生理功能。有趣的是，尽管细胞培养条件促进了未分化状态的维持，但神经分化还是发生了。这些结果表明，这五个 TF 中的每一个的过度表达都可以覆盖多能性特异性基因网络，并迫使 hESC 分化为神经元。 (C) 2017 Elsevier Inc. 保留所有权利。
Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROGI, NEUROG2, NEUROG3, NEURODI, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons. To this end, we established hESC lines in which each of these TFs can be overexpressed by the doxycycline-inducible piggyBac vector. The over expression of any of these five TFs indeed caused a rapid and rather uniform differentiation of hESCs, which were identified as neurons based on their morphologies, qRT-PCR, and immunohistochemistry. Furthermore, calcium-imaging analyses and patch clamp recordings demonstrated that these differentiated cells are electrophysiologically functional. Interestingly, neural differentiations occurred despite the cell culture conditions that rather promote the maintenance of the undifferentiated state. These results indicate that over-expression of each of these five TFs can override the pluripotency-specific gene network and force hESCs to differentiate into neurons. (C) 2017 Elsevier Inc. All rights reserved.