Franka Messner1, Marco Thurner1,2, Stefan Schneeberger3,4, Theresa Hautz5, Jule Müller1, Michael Blumer6, Julia Hofmann1, Rainer Marksteiner2, Sebastien Couillard-Despres7,8, Jakob Troppmair1, Dietmar Öfner1. 1. 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. 2. Innovacell Biotechnologie AG, Innsbruck, Austria. 3. 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. Stefan.Schneeberger@i-med.ac.at. 4. Department of Visceral, Transplant and Thoracic Surgery, Center of Operative Medicine, Medical University of Innsbruck, Anichstrasse 35, 6020, Innsbruck, Austria. Stefan.Schneeberger@i-med.ac.at. 5. 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. Theresa.Hautz@i-med.ac.at. 6. Department of Anatomy, Histology and Embryology, Division of Clinical and Functional Anatomy, Medical University of Innsbruck, Innsbruck, Austria. 7. Institute of Experimental Neuroregeneration, Spinal Cord Injury and Tissue Regeneration, Center Salzburg (SCI-TReCS), Paracelsus Medical University, Salzburg, Austria. 8. Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Abstract
BACKGROUND: 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. METHODS: 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 × 106 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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND: 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. METHODS: 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 × 106 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. RESULTS: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. CONCLUSION: 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.
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