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 repe… Show more
“…5 D, bottom). In summary, our results confirmed previous observations that modified IVT mRNA does not activate intracellular pattern recognition pathways 34 . Figure 5 Expression and functionality of NTCP protein dependent on the dose of ICT mRNA.…”
Infection of hepatocytes by hepatitis B virus (HBV) depends on surface expression of its receptor Na+-taurocholate-cotransporting polypeptide (NTCP), but sufficient NTCP expression is lacking in most cell lines. NTCP can be introduced by plasmid transfection or transduction by viral vectors to render cells permissive for HBV. However, transient transfection of hepatocyte-derived cell lines is inefficient, resulting in inhomogeneous protein expression and does not allow to adapt the level of NTCP expression. We therefore utilized in vitro transcribed mRNA to introduce NTCP into cells. Optimization using alternative cap structures and nucleotide modifications rendered mRNA transfection into different non-hepatic and hepatic cell lines very efficient. After transfection of mRNA, surface expression and functionality of NTCP was demonstrated by staining with an N-terminal HBV-preS peptide and bile acid uptake. Introduction of NTCP by mRNA transfection increased susceptibility of hepatoma cells to HBV in a dose-dependent manner. Transfection of NTCP mRNA into non-liver cells, in contrast, supported bile acid uptake but did still not render the cells permissive for HBV, demonstrating the requirement for additional host factors. Introduction of candidate host factors by mRNA transfection will allow for fast and convenient analysis of the viral life cycle using a transient, but reliable expression system.
“…5 D, bottom). In summary, our results confirmed previous observations that modified IVT mRNA does not activate intracellular pattern recognition pathways 34 . Figure 5 Expression and functionality of NTCP protein dependent on the dose of ICT mRNA.…”
Infection of hepatocytes by hepatitis B virus (HBV) depends on surface expression of its receptor Na+-taurocholate-cotransporting polypeptide (NTCP), but sufficient NTCP expression is lacking in most cell lines. NTCP can be introduced by plasmid transfection or transduction by viral vectors to render cells permissive for HBV. However, transient transfection of hepatocyte-derived cell lines is inefficient, resulting in inhomogeneous protein expression and does not allow to adapt the level of NTCP expression. We therefore utilized in vitro transcribed mRNA to introduce NTCP into cells. Optimization using alternative cap structures and nucleotide modifications rendered mRNA transfection into different non-hepatic and hepatic cell lines very efficient. After transfection of mRNA, surface expression and functionality of NTCP was demonstrated by staining with an N-terminal HBV-preS peptide and bile acid uptake. Introduction of NTCP by mRNA transfection increased susceptibility of hepatoma cells to HBV in a dose-dependent manner. Transfection of NTCP mRNA into non-liver cells, in contrast, supported bile acid uptake but did still not render the cells permissive for HBV, demonstrating the requirement for additional host factors. Introduction of candidate host factors by mRNA transfection will allow for fast and convenient analysis of the viral life cycle using a transient, but reliable expression system.
“…One possibility is suggested by reports that activation of antiviral or pro-inflammatory pathways, which still occurs at a low level even when countermeasures like modified mRNA and B18R are employed, might actually facilitate reprogramming. [38][39][40][41] If this idea is valid, the window in which such benign effects outweigh the cytotoxic and cytostatic effects of innate immune stimulation must be quite narrow. It also seems possible that collective effects involving cellcell communication are at work, linked to the homogeneity of factor expression achieved in the mRNA system-a speculative hypothesis prompted by the lockstep, population-wide phenotypic shifts characteristic of mRNA reprogramming cultures.…”
The discovery that ordinary skin cells can be turned into pluripotent stem cells by the forced expression of defined factors has raised hopes that personalized regenerative treatments based on immunologically compatible material derived from a patient's own cells might be realized in the not-too-distant future. A major barrier to the clinical use of induced pluripotent stem cells (iPSCs) was initially presented by the need to employ integrating viral vectors to express the factors that induce an embryonic gene expression profile, which entails potentially oncogenic alteration of the normal genome. Several "non-integrating" reprogramming systems have been developed over the last decade to address this problem. Among these techniques, mRNA reprogramming is the most unambiguously "footprint-free," most productive, and perhaps the best suited to clinical production of stem cells. Herein, we discuss the origins of the mRNA-based reprogramming system, its benefits and drawbacks, recent technical improvements that simplify its application, and the status of current efforts to industrialize this approach to mass-produce human stem cells for the clinic.Just over a decade has elapsed since the publication of Shinya Yamanaka's groundbreaking work 1 showing that ordinary skin cells can be "reprogrammed" by the expression of a cocktail of transcription factors into induced pluripotent stem cells (iPSCs) capable of giving rise to any cell or tissue of the body. Already, the first regenerative therapies based on these cells, focused on age-related macular degeneration, ischemic heart disease, and Parkinson's disease, have progressed to the stage of clinical trials. 2 The advent of cellular reprogramming holds out the tantalizing prospect it might be possible to turn a patient's own cells into a limitless supply of physiologically rejuvenated, immunologically compatible stem cells that can be coaxed to become specialized cells, tissues, and organs for transplant back into the donor, enabling new treatments for a wide range of diseases and for the maladies of old age.Clearly, there is a long road ahead before this futuristic vision can be fully realized, with many technical, financial and regulatory obstacles to be overcome. A major hurdle to any clinical application of iPSCs made with Yamanaka's original method was its dependence on integrating viral gene expression vectors to effect reprogramming, as the resulting heritable changes to cellular DNA would entail an unacceptable risk of tumorigenicity were the iPSCs or their differentiated progeny to be transferred into a human host. The goal of achieving "footprint-free" reprogramming obsessed the field for several years in the wake of Yamanaka's breakthrough. This once-daunting challenge can now be considered a solved problem. One of the first compelling solutions presented relies on the sustained delivery of synthetic mRNA encoding Yamanaka's reprogramming factors. Today, mRNA reprogramming vies with other well-established "non-integrating" methods, but it remains one of the mo...
“…Although a very low level of inflammation may occur when modRNA and B18R were applied, this might facilitate reprogramming. Reports showed that deliberate control of TLR signaling-mediated inflammation is critical in the success of mRNA-induced somatic reprogramming [ 122 , 123 , 124 , 125 ].…”
Modified mRNA (modRNA)-based somatic reprogramming is an effective and safe approach that overcomes the genomic mutation risk caused by viral integrative methods. It has improved the disadvantages of conventional mRNA and has better stability and immunogenicity. The modRNA molecules encoding multiple pluripotent factors have been applied successfully in reprogramming somatic cells such as fibroblasts, mesenchymal stem cells, and amniotic fluid stem cells to generate pluripotent stem cells (iPSCs). Moreover, it also can be directly used in the terminal differentiation of stem cells and fibroblasts into functional therapeutic cells, which exhibit great promise in disease modeling, drug screening, cell transplantation therapy, and regenerative medicine. In this review, we summarized the reprogramming applications of modified mRNA in iPSC generation and therapeutic applications of functionally differentiated cells.
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