Improving the Footprint-Free iPSC Generation by Nonintegrative Self-Replicating RNA-Based Reprogramming

Objectives: In the fields of tissue engineering, regenerative medicine, and disease modeling the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is gaining in importance. The footprint-free generated patient-specific iPSCs have a tremendous potential as a cell source for the creation of desired types of autologous cells. In this study, the reprogramming of human fibroblasts using synthetic modified messenger RNAs (mRNAs) or self-replicating RNAs (srRNAs) encoding the reprogramming factors was compared.

Methods: Human fibroblasts were reprogrammed using 1.2 μg mRNA cocktail or 1 μg srRNA. The pluripotency was characterized by immunostaining and flow cytometry. The tree-lineage differentiation potential was analyzed in vitro, as well as in vivo, using CAM assay.

Results: Both RNA-based methods resulted in generation of integration-free iPSCs without genomic alterations. The in vitro pluripotency characteristics and in vivo trilineage differentiation capacity were comparable. However, the reprogramming of cells by transfection of an srRNA encoding all reprogramming factors was faster, less time-consuming, and more efficient than the daily transfection of cells with a cocktail of synthetic mRNAs encoding reprogramming factors.

Conclusion: This study demonstrated that the transfection of somatic cells with srRNA can result in more efficient generation of patient-specific iPSCs for future clinical applications in the field of regenerative medicine.