title: Differential effects of toll-like receptor stimulation on mRNA-driven myogenic conversion of human and mouse fibroblasts.
abstract: 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 RNA-sensing 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-β 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-β 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-β 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.
authors: Department of Surgery, Duke University, USA; Duke Translational Research Institute, Duke University Medical Center, Durham, NC 27710, USA. Electronic address: jaewoo.lee@duke.edu.
authors: Jaewoo
authors: J
authors: Lee
authors: Department of Surgery, Duke University, USA.
authors: Li
authors: L
authors: Xu
authors: Department of Biomedical Engineering, Duke University, USA.
authors: Tyler M
authors: TM
authors: Gibson
authors: Department of Biomedical Engineering, Duke University, USA.
authors: Charles A
authors: CA
authors: Gersbach
authors: Department of Surgery, Duke University, USA; Duke Translational Research Institute, Duke University Medical Center, Durham, NC 27710, USA. Electronic address: bruce.sullenger@duke.edu.
authors: Bruce A
authors: BA
authors: Sullenger
mesh_terms: D000818:Animals; D016923:Cell Death; D018274:Electroporation; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D016899:Interferon-beta / Q000378:metabolism; D051379:Mice; D024510:Muscle Development / Q000235:genetics; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism; D019899:Paracrine Communication; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051196:Toll-Like Receptor 3 / Q000378:metabolism*; D051199:Toll-Like Receptor 7 / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D017570:MyoD Protein; D012333:RNA, Messenger; D051196:Toll-Like Receptor 3; D051199:Toll-Like Receptor 7; D016899:Interferon-beta
keywords: Interferon; Reprogramming; Toll-like receptor; Transdifferentiation; Transfection; mRNA