Enhanced Angiogenesis and Reendothelialization by Mesenchymal Stem Cells Secreting Growth Factors Produced by Genome Editing

Abstract

Vessel is an important network in body, and it has various types ranging from the aorta and the capillary. Diseases in these vessel are critical and difficult to cure fundamentally. To overcome this huddle, the fundamental way to treat these diseases has been established by regenerating blood vessels with the combination of stem cell therapy and gene therapy. Mesenchymal stem cells enhance the regeneration of blood vessels, but their survival rate and cytokine secretion in an injected site are limited. Therefore, another approaches to solve these problems require. To address this, Hepatocyte Growth Factor (HGF) gene that facilitates vascular regeneration was integrated into the safe-harbor site in the chromosome of mesenchymal stem cells (MSCs) using the TALEN system. The Tet-on system was also used to control the expression of HGF. The expression regulated by Doxycyclin was successfully validated in protein level after the integration into the host chromosome. Inducible HGF expressing MSCs promoted migration in the short term, prevented cell death and promoted angiogenesis in the long term. Also, to actually evaluate the vessel regeneration ability of functional MSCs, it was encapsulated in RGD-alginate microgel and applied to the limb ischemia model, which includes a phenotype of a peripheral vascular disease. As a result, the regeneration of blood vessels was improved by the injection of HGF secreting MSCs. It proved that the inducible HGF-secreting MSCs are a valuable therapeutic tool for the treatment of vascular diseases that critically requires angiogenesis. <br/> Based on these therapeutic effects, it was also applied to the large animal model with aortic disease. Atherosclerosis is a fatal disease and is very difficult to treat. Although stents are used to treat these problems, there are still limitations such as restenosis and stent thrombosis. In order to solve the existing problems, the rapid reendothelialization should be induced after the stent transplantation. Thus, to induce reendothelialization, the stent was combined with stem cells that secrete HGF and vascular endothelial growth factor (VEGF), Known as a strong vessel induction factor, in an inducible manner. <br/> Angiogenic growth factor secreting MSCs were adhered to a stent coated with polydopamin, fibronectin and extracellular matrix. The expression of HGF and VEGF on the stent was confirmed. Furthermore, cell growth on stent was confirmed while maintaining their original characteristics. When transplanted into pigs, VEGF reduced restenosis in the short term, but excessive restenosis was observed after 2 weeks-time point. In the case of HGF, the restenosis tended to decrease even after 2 weeks, and the micro CT showed a flattened endothelial layer. The mixed condition of HGF and VEGF expressing MSCs at the ratio of 5:1 highly reduced the restenosis after 4 weeks of transplantation. Reendothelialization was accelerated in the HGF and VEGF 5: 1 groups, confirmed by immunohistochemistry. We also found the human cell population that were injected with the stent around a porcine cardiac vein. These results suggest that stem cells expressing HGF and VEGF in the stent promote reendothelialization and reduce restenosis in swine models. In conclusion, it is possible to promote vascular regeneration and highlighted the possibility of treatment using the stem cells with the regulated expression of angiogenic growth factors.<br/>