The therapeutic effects of MSCs-based IGF-1 EGFP dual gene by sorting system in rat myocardial infarction model

Abstract

Myocardial infarction (MI) is the most important disease of the cardiovascular diseases because it causes cardiomyocyte death, scar formation, wall thinning and collagen degradation via blockage of the coronary arteries. Mesenchymal stem cells (MSCs)-based gene therapy has attracted attention due to their unique therapeutic properties such as capacity of multipotent differentiation into variety of cell types, immunomodulation, and paracrine effects. Furthermore, Insulin-like growth factor-1 (IGF-1) has substantially beneficial effects on cardiomyocyte function including cell proliferation and survival. In the present study, a simplified method have been established the MSCs-based IGF-1/EGFP gene therapy using the selection plasmid vector with sorting system.

To improve the transfection efficiency of MSCs using non-viral carriers, the selection vector was constructed by inserting IGF-1 coding sequences as a target gene into pEF1/His C vector, and sequentially cloned enhanced green fluorescent protein (EGFP) as a selection marker gene with its CMV promoter. By using the selection vector (pEF1/His C::IGF-1::EGFP), both IGF-1 and EGFP genes were expressed in MSCs in vitro. Dual-genes expressing MSCs were collected solely via fluorescence activated cell sorter (FACS) system and the purity was approximately 95.1%.

To investigate the therapeutic effects by sorted pEF1/His C::IGF-1::EGFP-MSCs, rat MI models were divided into 4 groups; PBS, control MSCs, unsorting MSCs and sorting MSCs. After 2 weeks after MSCs transplantation, the MI hearts were harvested. The green fluorescence as transfected MSCs was visualized in myocardium of ischemic left ventricle and observed in both unsorting and sorting groups. Interestingly, the IGF-1 transfected MSCs were significantly localized in sorting groups compared to unsorting groups. Also, the expression of IGF-1 was increased in sorting groups.

The TTC staining and the Masson’s trichrome staining were proceeded to evaluate the therapeutic effects of dual gene system with FACS through histological assessment. The infarct size by TTC staining was significantly decreased in sorting groups compared to other groups (sorting 5.74% vs. PBS 31.50%, Intact MSCs 12.61%, unsorting 10.42%). The fibrosis area of myocardium layer relatively decreased in sorting group compared to other groups.

The present study demonstrated that the expression of IGF-1 can attenuate the fibrosis and reduce the infarct size in rat MI models. Also, the selection plasmid vector with sorting system efficiently enhanced therapeutic effects in MSCs-based gene therapy. Furthermore, this selection plasmid vector together with sorting system could be a practical application in a wide range of diseases. Also, it could be used in cell-based gene therapy as a treatment tool, by simply replacing the gene of interest or based cell source.