Generation of healthy mice from gene-corrected disease-specific induced pluripotent stem cells.
Generation of healthy mice from gene-corrected disease-specific induced pluripotent stem cells.
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DOI：
10.1371/journal.pbio.1001099
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发表时间：
2011-07
期刊：
影响因子：
9.8
通讯作者：
Cantz T
中科院分区：
文献类型：
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作者：
Wu G;Liu N;Rittelmeyer I;Sharma AD;Sgodda M;Zaehres H;Bleidissel M;Greber B;Gentile L;Han DW;Rudolph C;Steinemann D;Schambach A;Ott M;Schöler HR;Cantz T
使用酪氨酸血症1型小鼠模型（富马酰乙酰乙酸水解酶[FAH]缺乏症; FAH −/−小鼠）作为孤儿疾病（如遗传性代谢性肝病）的范例，我们评估了成纤维细胞来源的FAH −/−诱导的多能干细胞（iPS细胞）作为基因校正的靶点，并结合四倍体胚胎互补方法。首先，在表征FAH −/− iPS细胞系之后，我们将FAH −/−-iPS细胞与四倍体胚胎聚合，并获得完全FAH −/−-iPS细胞衍生的小鼠，这些小鼠存活并表现出创始FAH −/−小鼠的表型。然后，我们使用第三代慢病毒载体将FAH cDNA导入FAH −/−-iPS细胞，以产生基因校正的iPS细胞。通过高分辨率阵列CGH分析，我们无法检测到这些细胞中的任何染色体改变，并且在它们与四倍体胚胎聚集后，我们获得了完全iPS细胞衍生的健康小鼠，其足月发育效率高达63.3%。基因校正通过这些小鼠的FAH阳性细胞在撤回拯救药物NTBC（2-（2-硝基-4-氟甲基苯甲酰基）-1，3-环己二酮）后的长期存活和扩增进行功能验证。此外，我们的研究结果表明，肝脏特异性启动子（转甲状腺素蛋白，TTR）驱动的FAH转基因和强病毒启动子（来自脾病灶形成病毒，SFFV）驱动的FAH转基因挽救了来自各自基因校正的iPS细胞的小鼠中的FAH缺陷表型。总之，我们的数据表明，慢病毒基因修复策略不会消除成纤维细胞衍生的iPS细胞的全部多能潜能，并且iPS细胞的遗传操作与四倍体胚胎聚集相结合提供了一种实用且快速的方法来评估小鼠模型中人类疾病的基因校正的功效。多能干细胞具有无限的自我更新能力和分化成身体几乎所有细胞类型的潜力。因此，多能干细胞对未来的细胞疗法非常感兴趣，并且今天已经用于“在培养皿中”研究疾病和筛选新药。在开创性地发现诱导多能干细胞（iPS细胞）可以通过将四种转录因子递送到非多能细胞中来产生之后，人们对可以获得患者来源的多能干细胞并进行遗传校正以开发再生医学的定制疗法的想法产生了极大的热情。在这里，我们提出了一个急性代谢性肝功能衰竭的小鼠模型，满足这些标准。首先，我们通过严格的试验证明，疾病特异性iPS细胞表现出完整的细胞和发育潜力，iPS细胞衍生的小鼠忠实地复制了FAH −/−小鼠的表型。然后，我们通过慢病毒递送完整的基因拷贝来遗传修复疾病特异性iPS细胞，并研究了这种额外的遗传操作对这些细胞的影响。通过我们的分析，我们排除了基因校正的iPS细胞中的主要甚至次要的染色体畸变。最重要的是，我们证明了基因校正的细胞保持了它们的全部潜力，我们通过四倍体互补方法产生了完全来自这些修复细胞的活小鼠，这些小鼠是健康的，没有任何代谢性肝病的迹象。
Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH −/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH −/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH −/− iPS cell lines, we aggregated FAH −/−-iPS cells with tetraploid embryos and obtained entirely FAH −/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH −/− mice. Then, we transduced FAH cDNA into the FAH −/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models. Pluripotent stem cells have unlimited self-renewing capability and the potential to differentiate into virtually all cell types of the body. Pluripotent stem cells are therefore of great interest for future cell-based therapies and are already in use today for studying diseases “in a dish” and screening for new drugs. After the seminal discovery that induced pluripotent stem cells (iPS cells) can be generated by the delivery of four transcription factors into non-pluripotent cells, a tremendous amount of enthusiasm arose about the idea that patient-derived pluripotent stem cells could be obtained and genetically corrected in order to develop customized therapies for regenerative medicine. Here, we present a mouse model of acute metabolic liver failure that fulfills such criteria. First, we demonstrated by stringent assays that disease-specific iPS cells exhibited full cellular and developmental potential and the iPS cell–derived mice reproduced the phenotypes of the founding FAH −/− mice faithfully. Then, we genetically repaired the disease-specific iPS cells by lentiviral delivery of an intact gene copy, and we investigated the impact of this additional genetic manipulation on these cells. With our analyses, we ruled out major, and even minor, chromosomal aberrations in the gene-corrected iPS cells. Most importantly, we demonstrated that the gene-corrected cells maintained their full potential and we generated viable mice that were completely derived from these repaired cells via tetraploid complementation approach, and these mice were healthy, without any signs of the metabolic liver disease.
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影响因子：
46.9
作者：
Azuma, Hisaya;Paulk, Nicole;Grompe, Markus
通讯作者：
Grompe, Markus
影响因子：
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作者：
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通讯作者：
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64.8
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