Direct Reprogramming of Fibroblasts into Functional Cardiomyocyte-like Cells without Viral Integration

Abstract

We and others reported that cardiomyocyte-like cells (iCMs) can be directly generated from mouse and human fibrob lasts by viral overexpression of the cardiac transcription factors, including Gata4, Mef2c, and Tbx5. This new strategy may have great potential for regenerative purposes, but a major limitation is the use of potentially harmful genome-integrating viruses. As an important translational step towards clinical therapy, we investigated whether we can reprogram fibroblasts into functional iCMs using non-integrating Sendai viruses (SeV), which transiently express cardiac reprogramming factors. Multiple cardiac genes were induced and fibroblast genes were suppressed in fibroblasts transduced with SeV, as shown by quantitative RT-PCR. FACS analysis demonstrated that αMHC-GFP reporter and cardiac troponin T (cTnT) protein were induced by SeV transduction. Immunohistochemistry revealed that the SeV-transduced cells expressed α-actinin, cTnT, and ANP after 2 weeks of transduction, and the cells had clear sarcomeric structures. Functionally, a subset of the iCMs started to contract spontaneously after 4 weeks in culture. Importantly, SeV transgene did not integrate into the iCMs genome. Thus, our results provide strong evidence that insertional mutagenesis is not required for direct cardiac reprogramming. Sendai viral reprogramming may provide an improved method for generating iCMs, which may facilitate future applications of the direct cardiac reprogramming towards clinical purposes.