Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines.
Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines.
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DOI：
10.1089/scd.2011.0688
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发表时间：
2012-08
影响因子：
4
通讯作者：
C. Zou;Bin-Kuan Chou;Sarah N. Dowey;K. Tsang;Xiaosong Huang;Cyndi F. Liu;Cory J. Smith;J. Yen
中科院分区：
文献类型：
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作者：
C. Zou;Bin-Kuan Chou;Sarah N. Dowey;K. Tsang;Xiaosong Huang;Cyndi F. Liu;Cory J. Smith;J. Yen
从体细胞类型诱导的多能干细胞 (iPSC) 的衍生以及随后对疾病特异性或患者特异性 iPSC 进行基因修饰是其在疾病建模以及未来细胞和基因治疗应用中的关键步骤。这些过程的常规程序需要与原代小鼠胚胎成纤维细胞 (MEF) 共培养，以支持人类 iPSC 以及胚胎干细胞 (ESC) 的自我更新和克隆生长。然而，MEF 质量的可变性会影响所有这些步骤的效率。此外，动物来源的饲养者可能会阻碍人类干细胞的临床应用。为了克服这些障碍，我们通过在成体间充质干细胞中稳定表达人端粒酶逆转录酶、Wnt3a和耐药基因来建立永生化的人饲养细胞系。在这里，我们证明这些永生化的人类饲养细胞支持有效衍生无病毒、无整合的人类 iPSC 以及人类 iPSC 和 ESC 的长期扩增。此外，这些饲养层的耐药特性还支持由piggyBac DNA转座介导的非病毒基因高效转移和表达。重要的是，这些人类饲养细胞在支持人类 iPSC 中同源重组介导的基因靶向方面表现出优于 MEF 的能力，使我们能够有效地将转基因靶向最近衍生的免整合 iPSC 中的 AAVS1 安全港位点。我们的结果对人类 iPSC 的疾病建模和转化应用具有重大意义，因为这些工程化人类细胞系为基因修饰提供了更有效的工具，并为支持人类 iPSC 和 ESC 的自我更新提供了更安全的替代方案。
Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.