GFP Labelled Sendai Virus

Direct cardiac reprogramming holds great promise for regenerative medicine. Direct reprogramming of induced cardiomyocyte-like cells (iCMs) has previously been generated using retroviral vectors overexpressing Gata4, Mef2c, and Tbx5 (GMTs). However, integrating vectors carry risks associated with insertional mutagenesis and disruption of gene expression, and are inefficient. Here, researchers show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram mouse and human fibroblasts into integration-free iCMs through robust transgene expression. SeV-GMTs generated 100-fold more beating iCMs than retroviral-GMTs and reduced the time to induce beating cells in mouse fibroblasts from 30 to 10 days. In vivo lineage tracing revealed that gene transfer with SeV-GMTs was more efficient than retroviral-GMTs in reprogramming resident cardiac fibroblasts into iCMs in infarcted mouse hearts. Furthermore, SeV-GMTs improved cardiac function and reduced fibrosis after myocardial infarction. Therefore, the highly efficient, nonintegrating SeV vector serves as a powerful system for cardiac regeneration.

Here, the researchers tested whether gene transfer of SeV-GMT into infarcted hearts could reprogram resident CFs into iCMs in vivo. They injected SeV-GFP (GFP Labelled Sendai Virus) directly into infarcted hearts of ICR mice immediately after coronary artery ligation (Figure 1A). One week later, SeV-GFP efficiently infected the infarct and border zones, but not the distal regions (Figures 1A and 1B). IHC showed that SeV-GFP+ cells in the infarct border zone expressed vimentin and collagen 1, markers of non-myocytes and fibroblasts, but not α-actinin (Figures 1B-1D). These results suggest that SeV vectors specifically infect non-cardiomyocytes (mainly resident CFs) in infarcted hearts of mice, similar to retroviral pMX vectors.

Figure 1. Lineage Tracing Demonstrates Efficient In Vivo Cardiac Reprogramming by SeV-GMT. (Miyamoto K, et al., 2018)

Next, the researchers determined whether SeV-GMT could reprogram resident CFs into iCMs in vivo using fibroblast lineage tracing mice. They crossed Tcf21^iCre knock-in mice with R26R-tdTomato reporter mice to generate Tcf21^iCre/R26-tdTomato fibroblast lineage tracing mice (Figure 1E). Tcf21^iCre/R26-tdTomato mice were treated with tamoxifen for 5 days to mark cells expressing TCF21, and coronary artery ligation was performed 7 days after the last tamoxifen treatment. The researchers found a large number of tdTomato+ fibroblasts within the infarct edge area 1 week after MI, and tdTomato+ cells did not express cTnT (Figure 1F and 1G). These results indicate that direct injection of SeV-GMT into mouse infarcted hearts can reprogram resident CFs into iCMs in vivo, and SeV-GMT improves the efficiency and quality of cardiac reprogramming in vivo compared with pMX-MGT, which is consistent with the in vitro results (Figure 1H-1I).