Abstract 12423: Rapid And Robust Reprogramming Of Fibroblasts Into Integration-free Functional Cardiomyocytes With Sendai Viral Vectors
Rationale: Direct reprogramming of fibroblasts into functional cardiomyocytes (iCMs) holds great potentials for disease modeling and regenerative purposes, but the technique thus far has been slow and inefficient. Moreover, integration of viral transgenes into host genome may hinder clinical applications of this strategy.
Objective: The aim of this study is to develop new Sendai viral vectors (SeVs) which overexpress reprogramming factors without genomic integration, and to determine the efficiency and safety of cardiac reprograming with SeVs.
Methods and Results: We generated separate Gata4 (G), Mef2c (M), and Tbx5 (T) SeVs, and a polycistronic SeV containing GMT in a single transgene (SeV-GMT). Transduction of separate G, M, T SeVs did not induce cardiac reprogramming in mouse fibroblasts. In contrast, the polycistronic SeV-GMT greatly promoted cardiac reprogramming and induced spontaneously beating iCMs up to 40% of the starting fibroblasts, which was 100-fold greater than the retroviral-mediated reprogramming. Moreover, SeV-GMT shortened the duration to induce beating cells from 30 to 10 days, compared to retrovirus-GMT, demonstrating rapid and robust cardiac induction with SeV-GMT. FACS analyses demonstrated that the number of cardiac troponin T (cTnT) + cells and the intensity of cTnT expression were strongly increased with SeV-GMT compared with retrovirus-GMT. EdU incorporation assays and sequential gene expression profiles revealed that the increase in beating iCMs with SeV-GMT was not due to cell proliferation or conversion to multipotent cardiac progenitor cells. SeV-GMT transduction resulted in lower expression of Tbx5 compared to separate G, M, T SeV transduction or retroviral-GMT transduction. Supplementation of Tbx5 in SeV-GMT-transduced cells greatly reduced cardiac reprogramming, suggesting stoichiometry of transcription factors might be optimal in SeV-GMT. Genomic DNA analyses revealed that the SeV transgenes were not integrated into the host genome, and no tumors were detected in culture after 2 months of SeV-GMT transduction.
Conclusions: We developed a highly efficient and safe direct cardiac reprogramming platform with SeV-GMT.
Author Disclosures: K. Miyamoto: None. H. Y amakawa: None. N. Muraoka: None. T. Sadahiro: None. H. Kojima: None. S. Haginiwa: None. M. Isomi: None. Y. Kuishi: None. S. Ninomiya: None. K. Fukuda: None. M. Ieda: None.
- © 2015 by American Heart Association, Inc.