The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts
The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts
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DOI：
10.1016/j.biomaterials.2012.02.025
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发表时间：
2012-06-01
期刊：
影响因子：
14
通讯作者：
Adjaye, James
中科院分区：
文献类型：
--
作者：
Drews, Katharina;Tavernier, Geertrui;Adjaye, James
在体细胞中通过脂质转染递送编码重编程因子的mRNA是用于衍生无整合iPSC的理想方法。然而，缺乏可重复性意味着在该方案被普遍接受之前还需要克服重大障碍。这项研究证明了我们内部合成的mRNA在人成纤维细胞核内表达重编程因子（OCT 4、SOX 2、KLF 4、c-MYC）的功能。然而，在重复转染后，如MTT细胞毒性测定所示，mRNA诱导细胞活力的严重丧失。微阵列转录组数据显示，细胞存活率低主要是由于外源性mRNA引发的先天免疫反应。我们通过定量实时PCR验证了mRNA转染对关键免疫应答相关转录物水平的影响，包括IFNB 1，RIG-I，PKR，IL 12 A，IRF 7和CCL 5，并将这些与先前发表的介导体细胞重编程的其他方法诱导的水平直接进行比较。最后，我们评估了化合物（B18 R、氯喹、TSA、Pepinh-TRIF、Pepinh-MYD），这些化合物以其抑制细胞先天免疫应答的能力而闻名。然而，这些都没有达到预期的效果。这里提供的数据应该为进一步研究其他免疫抑制策略提供基础，这些策略可能有助于人类细胞中有效的mRNA介导的细胞重编程。(c)2012爱思唯尔有限公司保留所有权利。
Delivery of reprogramming factor-encoding mRNAs by means of lipofection in somatic cells is a desirable method for deriving integration-free iPSCs. However, the lack of reproducibility implies there are major hurdles to overcome before this protocol becomes universally accepted. This study demonstrates the functionality of our in-house synthesized mRNAs expressing the reprogramming factors (OCT4, SOX2, KLF4, c-MYC) within the nucleus of human fibroblasts. However, upon repeated transfections, the mRNAs induced severe loss of cell viability as demonstrated by MTT cytotoxicity assays. Microarray-derived transcriptome data revealed that the poor cell survival was mainly due to the innate immune response triggered by the exogenous mRNAs. We validated the influence of mRNA transfection on key immune response-associated transcript levels, including IFNB1, RIG-I, PKR, IL12A, IRF7 and CCL5, by quantitative real-time PCR and directly compared these with the levels induced by other methods previously published to mediate reprogramming in somatic cells. Finally, we evaluated chemical compounds (B18R, chloroquine, TSA, Pepinh-TRIF, Pepinh-MYD), known for their ability to suppress cellular innate immune responses. However, none of these had the desired effect. The data presented here should provide the basis for further investigations into other immunosuppressing strategies that might facilitate efficient mRNA-mediated cellular reprogramming in human cells. (c) 2012 Elsevier Ltd. All rights reserved.