Department of Orthopedic Surgery
University Hospital of Geneva and Faculty of Medicine
Improved survival and better engraftment of human myogenic reserve cells, as compare to human myoblasts, after intramuscular transplantation in immunodeficient mice
Thomas Laumonier1, Flavien Bermont1, Pierre Hoffmeyer1, Jacques Menetrey1.
1Department of Orthopedic Surgery, University Hospital and Faculty of Medicine, Geneva, Switzerland
Introduction: Satellite cells are Pax7+ muscle stem cells that are essential for skeletal muscle growth and regeneration. They are, therefore, a potential stem cell source to treat muscle disorders occurring after massive muscle injuries or muscular dystrophies. Nevertheless, satellite cells are difficult to expand in vitro without dramatically reducing their regenerative potential. In the present study, we investigate if human myogenic reserve cells (hMRC), defined as a population of quiescent myogenic stem cell, may represent a good source of progenitor cells for use in muscle repair applications.
Methods: Procedures undertaken with human samples and mice were in compliance with the national and international guidelines. After isolation of human myoblasts by flow cytometry (100% CD56+), cells were expanded and place under differentiation medium (DM) conditions that promote generation of hMRC. Cells were characterized, transduced with a lentiviral vector encoding for the renilla luciferase bioluminescence marker gene and transplanted in injured muscles of immunodeficient mice. Cell survival was evaluated at day 0, 4, 7, 14 and 21 after injection using non-invasive bioluminescent techniques. Mice were then sacrificed and skeletal muscles prepared for histological and immunohistological analysis.
Results: Freshly isolated human myoblasts were expanded in growth medium (GM) and switched to DM for 2 days. Fusion indexes were quantified and 63 ± 9.5% of the nuclei were inside myotubes (n=6). The nature of the mononucleated non-fusing cells (37 ± 9.5%), defined as hMRC, was examined. We determined that 80% of hMRC showed similarities with human quiescent satellite cells (CD56+/Pax7+/MyoD-/Ki67- cells). hMRC re-exposed to GM and DM conditions were able to grow and to reform both myotubes and myogenic reserve cells. By western blot, we observed a 4-fold increase for Pax7 expression and a 10-fold decrease for MyoD expression, in hMRC as compare to human myoblasts. In vivo, we showed a significant improvement in the survival of hMRC as compare to human myoblasts at 4 days, 14 days and 21 days after transplantation in injured muscles (60.5± 5.8 % vs 34.1 ± 6.1% respectively, at day 21, p=0.005, n=8). At day 21, we observed, in both groups, the presence of human lamin A/C+ nuclei and human spectrin+ muscle fibers with no significant difference. Nevertheless, the ratio of Pax7+/human lamin A/C+ cells was improved by 20% after transplantation of hMRC as compare to human myoblasts.
Conclusions: Taken together, we provide evidence that hMRC generated in vitro, have similarities with human quiescent satellite cells. Survival of hMRC was improved as compare to human myoblasts and hMRC make a greater contribution to the satellite cell pool in host muscles. These data support the hypothesis that hMRC represent a promising source for improving regeneration of skeletal muscles.
15:30 - 17:00
|Islet Cell Transplantation - Miscellaneous||Improved survival and better engraftment of human myogenic reserve cells, as compare to human myoblasts, after intramuscular transplantation in immunodeficient mice||Room 111-112|