Published online before print July 31, 2008

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(American Journal of Pathology. 2008;173:781-791.)
© 2008 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2008.070902

Muscle CD31(–) CD45(–) Side Population Cells Promote Muscle Regeneration by Stimulating Proliferation and Migration of Myoblasts

Norio Motohashi^*, Akiyoshi Uezumi^*, Erica Yada^*, So-ichiro Fukada^*, Kazuhiro Fukushima^*¶, Kazuhiko Imaizumi, Yuko Miyagoe-Suzuki^* and Shin'ichi Takeda^*

From the Department of Molecular Therapy,^* National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo; the Division for Therapies against Intractable Diseases, Institute for Comprehensive Medical Science, Fujita Health University, Aichi; the Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka; the Laboratory of Physiological Sciences, Faculty of Human Sciences, Waseda University, Saitama; and the Third Department of Medicine, Neurology, and Rheumatology,^¶ Shinshu University School of Medicine, Matsumoto, Japan

CD31(–) CD45(–) side population (SP) cells are a minor SP subfraction that have mesenchymal stem cell-like properties in uninjured skeletal muscle but that can expand on muscle injury. To clarify the role of these SP cells in muscle regeneration, we injected green fluorescent protein (GFP)-positive myoblasts with or without CD31(–) CD45(–) SP cells into the tibialis anterior muscles of immunodeficient NOD/scid mice or dystrophin-deficient mdx mice. More GFP-positive fibers were formed after co-transplantation than after transplantation of GFP-positive myoblasts alone in both mdx and NOD/scid muscles. Moreover, grafted myoblasts were more widely distributed after co-transplantation than after transplantation of myoblasts alone. Immunohistochemistry with anti-phosphorylated histone H3 antibody revealed that CD31(–) CD45(–) SP cells stimulated cell division of co-grafted myoblasts. Genome-wide gene expression analyses showed that these SP cells specifically express a variety of extracellular matrix proteins, membrane proteins, and cytokines. We also found that they express high levels of matrix metalloproteinase-2 mRNA and gelatinase activity. Furthermore, matrix metalloproteinase-2 derived from CD31(–) CD45(–) SP cells promoted migration of myoblasts in vivo. Our results suggest that CD31(–) CD45(–) SP cells support muscle regeneration by promoting proliferation and migration of myoblasts. Future studies to further define the molecular and cellular mechanisms of muscle regeneration will aid in the development of cell therapies for muscular dystrophy.