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  The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts

Drews, K., Tavernier, G., Demeester, J., Lehrach, H., de Smedt, S. C., Rejman, J., et al. (2012). The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts. Biomaterials, 33(16), 4059-4068. doi:10.1016/j.biomaterials.2012.02.025.

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 Creators:
Drews, K.1, Author              
Tavernier, G.2, Author              
Demeester, J., Author
Lehrach, H.2, Author              
de Smedt, S. C., Author
Rejman, J., Author
Adjaye, J.1, Author              
Affiliations:
1Molecular Embryology and Aging (James Adjaye), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, Berlin, Germany, ou_1479654              
2Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, Berlin, Germany, ou_1433550              

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Free keywords: Cell Survival Cells, Cultured Fibroblasts/*cytology/immunology/metabolism Green Fluorescent Proteins/metabolism Humans Immunity, Innate Liposomes RNA, Messenger/*genetics Real-Time Polymerase Chain Reaction Reverse Transcriptase Polymerase Chain Reaction Transfection
 Abstract: 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.

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Language(s): eng - English
 Dates: 2012-02-282012-06
 Publication Status: Published in print
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 Table of Contents: -
 Rev. Type: Peer
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Title: Biomaterials
Source Genre: Journal
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Publ. Info: Guildford, England : Elsevier
Pages: - Volume / Issue: 33 (16) Sequence Number: - Start / End Page: 4059 - 4068 Identifier: ISSN: 0142-9612
CoNE: https://pure.mpg.de/cone/journals/resource/954925472369