Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 478, Issue 3, Pages 1484-1490Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2016.08.159
Keywords
Toll-like receptor; Reprogramming; Transdifferentiation; mRNA; Transfection; Interferon
Categories
Funding
- Robertson Foundation
- NIH [DP2OD008586, T32GM008555]
- NSF [CBET-1151035]
- Directorate For Engineering
- Div Of Chem, Bioeng, Env, & Transp Sys [1151035] Funding Source: National Science Foundation
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Transfection with in vitro transcribed mRNAs is a safe and effective tool to convert somatic cells to any cell type of interest. One caveat of mRNA transfection is that mRNAs are recognized by multiple RNAsensing toll like receptors (TLRs). These TLRs can both promote and inhibit cellular reprogramming. We demonstrated that mRNA transfection stimulated TLR3 and TLR7 and induced cytotoxicity and IFN-beta expression in human and mouse fibroblasts. Furthermore, mRNA transfection induced paracrine inhibition of repeated mRNA transfection through type I IFNs. Modified mRNAs (mmRNAs) containing pseudouridine and 5-methycytosine reduced TLR stimulation, cytotoxicity and IFN-beta expression in fibroblasts. Repeated liposomal transfection with MyoD mmRNAs significantly enhanced myogenic conversion of human and mouse fibroblasts compared with repeated transfection with MyoD mRNAs. Interestingly, electroporation of mRNAs and mmRNAs completely abrogated cytotoxicity and IFN-beta expression and also abolished myogenic conversion of fibroblasts. At a low concentration, TLR7/8 agonist R848 enhanced MyoD mmRNA-driven conversion of human fibroblasts into skeletal muscle cells, whereas high concentrations of R848 inhibited myogenic conversion of fibroblasts. Our study suggests that deliberate control of TLR signaling is a key factor in the success of mRNA-driven cellular reprogramming. (C) 2016 Elsevier Inc. All rights reserved.
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