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

Paper ID Volume ID Publish Year Pages File Format Full-Text
7293 544 2012 10 PDF Available
Title
The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts
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.

Keywords
mRNA; Reprogramming; Lipofection; Transfection; Immune response
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The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomaterials - Volume 33, Issue 16, June 2012, Pages 4059–4068
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us