Human skeletal muscle satellite cells are a manageable tool for gene therapy (P2.009)

OBJECTIVE To exploit the potential of human satellite cells for the development of therapies in muscle disorders. BACKGROUND Satellite cells, defined by the transcription factor Pax7, are the proper stem cells of skeletal muscle. They are indispensable for muscle regeneration. Most knowledge about satellite cells was gained from mouse experiments. We studied human satellite cells while maintaining their natural stem cell niche. DESIGN/METHODS Freshly isolated human muscle fiber fragments (HMFF) (n>900) from adult donors (n=80, 20-80 years of age) were manually dissected and placed in tissue culture. Some fragments were subjected to hypothermic treatment at 4°C for one to five weeks before incubation at 37°C. HMFF were analysed by immunohistochemistry for the presence of Pax7, Myf5, NCAM, desmin, BrdU positive cells after various timepoints. HMFF were also transplanted into irradiated anterior tibial muscle of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (NOD mice). Last, HMFF were transfected with the Sleeping Beauty transposon-mediated nonviral gene transfer system carrying the fluorescence protein Venus. RESULTS Independent of donor age, as few as two to four Pax7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of NOD mice resulted in robust engraftment of human myoblasts. Further, we found that hypothermic treatment resulted in pure myogenic cultures without contaminating fibroblasts and improved muscle regeneration and proper homing to the stem cell niche. Finally, we successfully transfected the Venus gene into cultured Pax7+ satellite cells. CONCLUSIONS Our results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these Pax7+ satellite cells have potential to be useful in gene therapy. Disclosure: Dr. Spuler has nothing to disclose. Dr. Marg has nothing to disclose. Dr. Escobar has nothing to disclose. Dr. Spuler has nothing to disclose. Dr. Izsvak has nothing to disclose. Dr. Birchmeier has nothing to disclose.