Daniel Swarovski Research Laboratory (DSL), Department of Visceral, Transplant and Thoracic Surgery (VTT), Center of Operative Medicine, Medical University of Innsbruck (MUI), Innrain 66, 6020, Innsbruck, Austria.
Innovacell Biotechnologie AG, Innsbruck, Austria.
Stem Cell Res Ther. 2021 Feb 24;12(1):146. doi: 10.1186/s13287-021-02208-w.
Muscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration.
A clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 10 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration.
2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI.
WIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.
肌肉严重受到缺血/再灌注损伤(IRI)的影响。静止的卫星细胞分化为成肌祖细胞(MPC)具有显著的再生潜力。在此,我们建立了一种局部应用 MPC 治疗小鼠后肢缺血/再灌注模型,以研究肌肉再生所需的细胞移植和分化。
建立夹闭小鼠(C57b/6J)后肢缺血模型,以诱导骨骼肌 IRI。在 2 小时(h)温热缺血时间(WIT)和再灌注后,从分离的卫星细胞中获得表达报告蛋白的 MPC(TdTomato 或 Luci-GFP,1×10^5 个细胞),并肌肉内注射。通过流式细胞术和分化实验分析 MPC 的表面标志物表达和分化潜能。通过体内生物发光成像和活检的组织病理学评估来定量细胞命运、移植和再生。
2h WIT 导致肌肉严重 IRI,损伤后 14 天内通过组织病理学观察到肌纤维再生。体内生物发光成像显示,在研究期间(75 天),2h WIT 动物和对照动物的 MPC 报告蛋白信号。生物发光信号在注射部位检测到,并随时间增加。术后第 2 和 14 天,分别在宿主组织中检测到表达 TdTomato 的 MPC 和肌纤维,表明注射的 MPC 分化为肌纤维。与未发生缺血的对照组相比,2h WIT 后报告蛋白信号更高,提示注射到 IRI 损伤肌肉中的 MPC 生长和/或移植增强,拮抗 IRI 引起的肌肉损伤。
WIT 诱导的肌肉 IRI 需要增加注射的 MPC 的数量以进行移植和持续存在,这表明细胞治疗可能拮抗 IRI。需要进一步研究以评估 MPC 在缺血肢体损伤中的再生能力和治疗优势。