Generation of iPSCs by Nonintegrative RNA-Based Reprogramming Techniques: Benefits of Self-Replicating RNA versus Synthetic mRNA

Abstract

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is gaining in importance in the fields of regenerative medicine, tissue engineering, and disease modeling. Patient-specific iPSCs have as an unlimited cell source a tremendous potential for generating various types of autologous cells. For the future clinical applicability of these iPSC-derived cells, the generation of iPSCs via nongenome integrating methods and the efficient reprogramming of patients’ somatic cells are required. In this study, 2 different RNA-based footprint-free methods for the generation of iPSCs were compared: the use of synthetic modified messenger RNAs (mRNAs) or self-replicating RNAs (srRNAs) encoding the reprogramming factors and GFP. Using both RNA-based methods, integration-free iPSCs without genomic alterations were obtained. The pluripotency characteristics identified by specific marker detection and the in vitro and in vivo trilineage differentiation capacity were comparable. Moreover, the incorporation of a GFP encoding sequence into the srRNA enabled a direct and convenient monitoring of the reprogramming procedure and the successful detection of srRNA translation in the transfected cells. Nevertheless, the use of a single srRNA to induce pluripotency was less time consuming, faster, and more efficient than the daily transfection of cells with synthetic mRNAs. Therefore, we believe that the srRNA-based approach might be more appropriate and efficient for the reprogramming of different types of somatic cells for clinical applications.

RNA-Based Strategies for Cardiac Reprogramming of Human Mesenchymal Stromal Cells

“…Hence, there is an urgent need for further optimization. Since mRNAs are degraded over time, multiple transfection steps might increase the reprogramming efficiency, a strategy that is already applied for the generation of iPSCs from adult cells [74,80]. Moreover, proportions of GATA4, MEF2C, and TBX5 protein expression has been described to play a crucial role for the quality of cardiac reprogramming [81], thus, different ratios of transfected mRNA could positively influence the outcome of reprogrammed adMSC.…”

Self-amplifying mRNA-Based Vaccine Technology and Its Mode of Action

“…To circumvent innate immune activation, lessons could be learned from viruses, as they have developed effective strategies to bypass innate immunity (Schulz and Mossman 2016). Co-expression of virally delivered IFN modulating proteins could be an effective solution for mRNA-based reprogramming approaches (Yoshioka and Dowdy 2017;Steinle 2019). To reduce PRR stimulation and consequent reduction of self-amplifying mRNA translation, Beissert et al have demonstrated that antigen expression from self-amplifying mRNA can be significantly improved in vitro and in vivo by co-transferring mRNAs encoding IFN and PKR inhibitory proteins, such as viral protein B18R, a decoy receptor of vaccinia virus, which binds type I IFNs (Beissert 2017).…”

Insulin/Glucose-Responsive Cells Derived from Induced Pluripotent Stem Cells: Disease Modeling and Treatment of Diabetes

“…Utilizing mRNA to express reprogramming factors has high efficiency, which can be even further improved by adding Lin28 to the Yamanaka reprogramming factors [ 33 ]. Culturing at 5% O 2 with addition of valproic acid in the medium also enhances efficiency [ 34 ].…”