Generation of Naivetropic Induced Pluripotent Stem Cells from Parkinson's Disease Patients for High-Efficiency Genetic Manipulation and Disease Modeling.
Generation of Naivetropic Induced Pluripotent Stem Cells from Parkinson's Disease Patients for High-Efficiency Genetic Manipulation and Disease Modeling.
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DOI：
10.1089/scd.2015.0079
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发表时间：
2015-11-01
影响因子：
4
通讯作者：
Feng J
中科院分区：
文献类型：
--
作者：
Hu Z;Pu J;Jiang H;Zhong P;Qiu J;Li F;Wang X;Zhang B;Yan Z;Feng J
Parkin 敲除啮齿类动物中缺乏稳健的帕金森病 (PD) 表型，以及在来自具有 Parkin 突变的 PD 患者的诱导多能干细胞 (iPSC) 的中脑 DA 神经元中鉴定出有缺陷的多巴胺能 (DA) 神经传递，证明了患者特异性 iPSC 作为有效系统来模拟 PD 中脑 DA 神经元的独特脆弱性的效用。为了研究帕金森病等疾病，人们在人类 iPSC 中开发高效的基因组工程技术方面付出了巨大的努力。在本研究中，我们通过 DOX 诱导的转基因（Oct4、Sox2、Klf4、c-Myc 和 Nanog）表达以及使用 2iL（MEK 抑制剂 PD0325901、GSK3 抑制剂 CHIR99021 和人 LIF）将患者特异性 iPSC 从引发状态转化为幼稚状态。这些患者特异性的幼稚 iPSC 在标记物表达、体外自发分化和体内畸胎瘤形成方面具有多能性。它们表现出与幼稚小鼠胚胎干细胞（ESC）非常相似的形态、增殖和克隆形成特征。幼稚型 iPSC 的高克隆效率和增殖率使得 GFP 通过类转录激活因子效应核酸酶非常有效地基因靶向 PITX3 位点。在撤回 DOX、2iL 并切换到引发状态 hESC 培养条件后，幼稚型 iPSC 可以很容易地恢复到引发状态。由恢复的 iPSC 分化而来的中脑 DA 神经元保留了 Parkin 突变引起的原始表型，证明了这些表型的稳健性以及基因组工程在患者特异性幼稚 iPSC 中用于研究 PD 的有用性。
The lack of robust Parkinson's disease (PD) phenotype in parkin knockout rodents and the identification of defective dopaminergic (DA) neurotransmission in midbrain DA neurons derived from induced pluripotent stem cells (iPSC) of PD patients with parkin mutations demonstrate the utility of patient-specific iPSCs as an effective system to model the unique vulnerabilities of midbrain DA neurons in PD. Significant efforts have been directed at developing efficient genomic engineering technologies in human iPSCs to study diseases such as PD. In the present study, we converted patient-specific iPSCs from the primed state to a naivetropic state by DOX-induced expression of transgenes (Oct4, Sox2, Klf4, c-Myc, and Nanog) and the use of 2iL (MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021, and human LIF). These patient-specific naivetropic iPSCs were pluripotent in terms of marker expression, spontaneous differentiation in vitro, and teratoma formation in vivo. They exhibited morphological, proliferative, and clonogenic characteristics very similar to naive mouse embryonic stem cells (ESC). The high clonal efficiency and proliferation rate of naivetropic iPSCs enabled very efficient gene targeting of GFP to the PITX3 locus by transcription activator-like effector nuclease. The naivetropic iPSCs could be readily reverted to the primed state upon the withdrawal of DOX, 2iL, and the switch to primed-state hESC culture conditions. Midbrain DA neurons differentiated from the reverted iPSCs retained the original phenotypes caused by parkin mutations, attesting to the robustness of these phenotypes and the usefulness of genomic engineering in patient-specific naivetropic iPSCs for studying PD.