Mesenchymal stem cells (MSCs) can improve limb perfusion and increase vessel density in a murine model of hindlimb ischemia. But low engraftment rate of those cells limited their therapeutic effect. Endothelial cells (ECs) play an important role in neovascularization. And MSCs can differentiate into ECs in vitro. The aim of this study is to investigate if EC differentiation of MSCs in vitro before transplantation is effective in improving therapeutic outcomes in the treatment of ischemic disease in a murine ischemia animal model. MSCs were isolated from the bone marrow of EGFP-transgenic mice by density gradient centrifugation. The identity of the MSCs was determined by their cluster of differentiation (CD) marker profile by flow cytometry. Inducing medium containing a few cytokines was applied to induce the MSCs to differentiate into ECs. Endothelial differentiation was quantitatively evaluated using flow cytometry, quantitative real-time PCR (qRT-PCR), immunofluorescence, Matrigel tube formation assay, and Dil-labeled acetylated low-density lipoprotein uptake assay. Mouse hindlimb ischemia model was made by excision of the femoral artery. Uninduced EGFP+ MSCs, induced EGFP+ MSCs, and PBS were intramuscularly injected into the gastrocnemius following ischemia no later than 24 h after operation. Restoration of blood flow and muscle function was evaluated by laser Doppler perfusion imaging. Immunofluorescence was conducted to evaluate the engraftment of transplanted MSCs. Histological analysis was performed to evaluate blood vessel formation. Induced EGFP+ MSCs expressed endothelial markers and exhibited tube formation capacity. Mice in the induced EGFP+ MSCs group had a better blood perfusion recovery, enhanced vessel densities, higher engraftment, and improved function of the ischemic limb than those in the uninduced EGFP+ MSCs or PBS groups. This study reveals that after short-term pre-treatment in the EC-inducing medium, induced MSCs acquire stronger vessel formation capability and enhanced angiogenic therapeutic effect in the murine hindlimb ischemia model.

中文翻译：

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在小鼠后肢缺血模型中，骨髓间充质干细胞来源的内皮细胞可增加毛细血管密度并加速血管生成。

在小鼠后肢缺血模型中，间充质干细胞（MSC）可以改善肢体灌注并增加血管密度。但是这些细胞的低植入率限制了它们的治疗效果。内皮细胞（EC）在新血管形成中起重要作用。MSC可以在体外分化为EC。这项研究的目的是研究在小鼠缺血动物模型中，移植前体外MSC的EC分化是否有效改善缺血性疾病的治疗效果。通过密度梯度离心从EGFP-转基因小鼠的骨髓中分离出MSC。通过流式细胞术通过它们的分化簇（CD）标记概况来确定MSC的身份。应用包含少量细胞因子的诱导培养基诱导MSC分化为EC。使用流式细胞仪，定量实时PCR（qRT-PCR），免疫荧光，Matrigel管形成测定和Dil标记的乙酰化低密度脂蛋白摄取测定对内皮分化进行定量评估。切除股动脉制成小鼠后肢缺血模型。缺血后不迟于手术后24小时将未诱导的EGFP + MSC，诱导的EGFP + MSC和PBS肌肉注射入腓肠肌。通过激光多普勒灌注成像评估血流和肌肉功能的恢复。进行了免疫荧光以评估移植的MSC的植入。进行组织学分析以评估血管形成。诱导的EGFP + MSC表达内皮标志物并显示出管形成能力。与未诱导的EGFP + MSCs或PBS组相比，诱导的EGFP + MSCs组的小鼠具有更好的血液灌注恢复，增强的血管密度，更高的植入率和缺血肢体的功能。这项研究表明，在EC诱导培养基中进行短期预处理后，诱导的MSC在小鼠后肢缺血模型中获得了更强的血管形成能力并增强了血管生成治疗作用。