Clonal Multipotency of Skeletal Muscle-derived Stem Cells Between Mesodermal and Ectodermal Lineage

¹ Muscle Physiology & Cell Biology Unit, Department of Regenerative Medicine, Division of Basic Clinical Science; Tokai University School of Medicine, 143-Shimokasuya, Isehara, Kanagawa 259-1143 Japan
² Muscle Physiology & Cell Biology Unit, Teaching & Research Support Center; Tokai University School of Medicine, 143-Shimokasuya, Isehara, Kanagawa 259-1143 Japan
³ Muscle Physiology & Cell Biology Unit, Department of Orthopedics, Division of Surgery; Tokai University School of Medicine, 143-Shimokasuya, Isehara, Kanagawa 259-1143 Japa
⁴ Department of Regenerative Medicine, Division of Basic Clinical Science; Tokai University School of Medicine, 143-Shimokasuya, Isehara, Kanagawa 259-1143 Japan
⁵ Muscle Physiology & Cell Biology Unit, Department of Urology, Division of Surgery, Tokai University School of Medicine, 143-Shimokasuya, Isehara, Kanagawa 259-1143 Japan
⁶ Muscle Physiology & Cell Biology Unit, Tokai University School of Medicine, 143-Shimokasuya, Isehara, Kanagawa 259-1143 Japan; Stem Cell Research, Research Center Japan, Nihon Schering K.K., 1-5-5 Minatojima-minami-machi, Chuo-ku, Kobe 650-0047 Japan

	Abstract

The differentiation potential of skeletal muscle-derived stem cells (MDSCs) after in vitro culture and in vivo transplantation has been extensively studied. However, the clonal multipotency of MDSCs has yet to be fully determined. Here, we show that single skeletal muscle-derived CD34^-/CD45^- (Sk-DN) cells exhibit clonal multipotency that can give rise to myogenic, vasculogenic and neural cell lineages after in vivo single cell-derived single sphere implantation and in vitro clonal single cell culture. Muscles from GFP transgenic mice were enzymatically dissociated and sorted based on CD34 and CD45. Sk-DN cells were clone-sorted into a 96-well plate and were cultured in collagen-based medium with bFGF and EGF for 14 days. Individual CFUs were then transplanted directly into severely damaged muscle together with 1x10⁵ competitive carrier Sk-DN cells obtained from wild-type mice muscle expanded for 5 days under the same culture conditions using 35-mm culture dishes. Four weeks after transplantation, implanted GFP⁺ cells demonstrated differentiation into endothelial, vascular smooth muscle, skeletal muscle and neural cell (Schwann cell) lineages. This multipotency was also confirmed by expression of mRNA markers for myogenic (MyoD, myf5), neural (Musashi-1, Nestin, NCAM1, PMP22, Nucleostemin) and vascular (-smooth muscle actin, smoothelin, VE-cadherin, TEK) stem cells by clonal (single-cell derived) single-sphere RT-PCR. About 70% of clonal CFUs exhibited expression of all three cell-lineages. These findings support the notion that Sk-DN cells are a useful tool for damaged muscle-related tissue reconstitution by synchronized vasculogenesis, myogenesis and neurogenesis.

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