379. Myogenic Differentiation of Multipotent Adult Progenitor Cells Derived from Bone Marrow and Muscle Tissue
Authors: Morayma Reyes, Sheng Li, En Kimura, Leonard Meuse, Jeffrey Chamberlain
Journal: Molecular Therapy (2004)

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Multipotent Adult Progenitor Cells, or MAPC, can be isolated from mouse bone marrow (BM), muscle and brain. MAPC can differentiate in vitro and in vivo into mesodermal, endodermal, and ectodermal tissue. We report here the isolation of MAPC from BM and muscle of mdx mice as well as wild type C57BL10 and C57BL6 mice. These MAPC from BM and muscle can be expanded for more than 50 cell doublings and can differentiate into endothelial cells, hepatocytes and neurons. MAPC are negative for satellite cell markers and negative for all the muscle regulatory factors, MRFs. Initial studies showed that the abundance of MAPC in mdx muscle is 10 folds lower as compared to wt muscle at 8 weeks of age. This decrease in abundance is augmented at older ages. Also MAPC from mdx muscle have a faster growth rate (cell doubling = 24 hrs) than MAPC from wt muscle (cell doubling = 36 hrs) and seemed more prone to differentiate into myoblasts as higher rate of spontaneous differentiation into myotubes (see below) was observed in mdx muscle MAPC as compared to wt muscle MAPC. This suggests that muscle MAPC may participate in muscle regeneration and thus are wasted in old mdx muscle. Studies to test this hypothesis are ongoing. When MAPC are induced with bFGF and 15% horse serum, a rapid upregulation of Myf5 is seen at d4 followed by transient expression of MyoD. At d7 expression of Myogenin and MRF4 is seen in cells undergoing cell fusion. After d10, more than 50% of the cells have formed myotubes. Myoblasts derived from these muscle MAPC can then be split for more than 10 passages (40 cell doublings). At every passage, myotube formation is seen together with proliferation of small cells that are Myf5+ but negative for all the other MRFs, indicating the persistence, maintenance and proliferation of a possible muscle stem cell in these culture conditions. This is the first demonstration that a non-myogenic adult stem cell undergoes myogenesis in a developmental stage-specific pattern. In contrast, when BM MAPC are induced with bFGF and 15% FCS, more than 50% cell death is observed in the first 7d. After d21, myotube formation is seen in the remaining cells. However, it seems that BM MAPC terminally differentiate into myotubes as they can not be passaged. This observation indicates that muscle MAPC, as compared to BM MAPC, can differentiate into myoblasts and form myotubes faster and more efficiently. Studies to determine if muscle MAPC have a predisposition or cellular memory to differentiate into the tissue of origin are ongoing. To prove that the myogenic potential of muscle MAPC is not due to satellite cell contamination, muscle MAPC were single cell cloned. 50% of the clones that could be successfully proliferated for more than 20 cell doublings were able to differentiate into skeletal myoblasts and other lineages such as hepatocytes, endothelium, neurons and skeletal myoblasts. MAPC can be transduced very efficiently obtaining more than 90% transduction frequency with a GFP containing lentivirus vector. Finally, when undifferentiated MAPC (transduced with a GFP-lentiviral vector) are intramuscularly transplanted in the anterior tibialis muscle of mdx mice, significant myofiber regeneration was seen.