Preclinical Evaluation of Bone Marrow Mesenchymal Stem Cells Overexpressing MicroRNA-126 to Treat Critical Limb Ischemia

Abstract

Introduction: Critical limb ischemia (CLI) considered as the most severe form of peripheral artery disease (PAD). Current therapy for CLI are surgical reconstruction and endovascular therapy or limb amputation (for patients with no treatment options). Neovasculogenesis induced by stem cells including mesenchymal stem cells (MSCs) therapy is a promising approach to treat CLI. But this method of treatment faces challenges such as: MSCs survival and paracrine secretion. MicroRNAs are post transcriptional regulatory molecules that regulate many biological processes including VEGF pathway. MicroRNAs could be used to increase viability and angiogenic potential of MSCs. This study was conducted to reinforce and increase the angiogenic potential of BM-MSCs by using microRNA-126 and evaluate the effect of this stem cell gene therapy on treatment of ischemic tissues in CLI mouse models. Methods: BM-MSCs were isolated from male C57 BL/6 inbred mice and characterized by morphology, flow cytometry, differentiation to osteocyte and adipocyte. Transformed BM-MSCs containing miR-126 were produced by using lentiviral vector. Then femoral artery ligation and total excision of the femoral artery was performed on C57BL/6 mice to create CLI model. Animals were allocated to control, BM-MSCs, virus and BM-MSCs miR-126 groups and defined number of the cells and virus were injected 24 h after surgery. In order to determine in vitro and in vivo effects, the following tests were performed: wound healing assay, behavioral tests including: Tarlov, Ischemia, Modified ischemia, Function and the grade of limb necrosis scores, donor cell survival assay, real-time PCR and histological analysis. Results: Results indicated that during 28 days after transplantation, BM-MSCs and virus groups had an enhancing effect on angiogenesis. BM-MSCs miR-126 group had remarkable effect on endothelial cell migration, muscle restructure, functional improvements and neovascularization in ischemic tissues and led to more effective treatment. In vivo evaluation showed that miR-126 could increase BM-MSCs survival and paracrine secretion of angiogenic factors such as VEGF, and led to remarkable functional improvements and neovascularization in ischemic tissues. Conclusions: According to the obtained results, it could concluded that combination of BM-MSCs and miR-126 leads to more effective recovery from critical limb ischemia compared to using them alone. In fact, miR-126 can be used as a strong modifier to reinforce the angiogenic potential of BM-MSCs, leading to more effective treatment for CLI.

Probing the effects of polysaccharide hydrogel composition on the viability and pro-angiogenic function of human adipose-derived stromal cells

“…In general, further research is critically needed to establish targets for a therapeutic cell dose, which may be different if the cells are delivered using a biomaterial versus saline. It is important to recognize that these targets will also depend on the specific MSC source applied, as well as the characteristics of the cell donor, including age, sex, and health status [58]- [60]. Overall, there is a need for the field to establish potency indicators for the specific cell population(s) of interest, to select donor cells that have the functional capacity to reliably and predictably stimulate regeneration and limit issues associated with cell donor variability [61].…”