Résumé :
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The last decade, multi-lineage stem cells encountered in various adult tissues have been described as exhibiting myogenic potential in experimental conditions. These stem cells include Side Population cells, mesoangioblasts, CD133+ progenitors and Muscle-Derived Stem Cells (MDSC). The last ones have been initially selected in mice as late-adherent cells, using preplating technique. Here, we focused on the canine MDSC isolation and their in vivo myogenic potential. Muscle-derived cells (MDC) were extracted from healthy dogs and submitted to classical preplating technique. MDSC were analyzed in primary and clonal cultures, and investigated for their cell cycle status, multi-lineage differentiation potential and phenotype. MDSC and myoblasts were transduced with nls LacZ retrovirus, then intra-muscularly injected in 4-month old GRMD dogs, whom muscles show massive fiber necrosis with endomysial and perimysial connective tissue proliferation. After 5 weeks, the injected muscles were dissected and histologically analyzed for the presence of ?-gal+ nuclei. We demonstrated that MDSC could be isolated in large animal model : MDSC represented 2.9% of all MDC. In addition to their proliferation/fusion behavior in vitro, MDSC displayed other stem cell features, including differentiation into adipogenic and osteogenic cells, G0-cell cycle arrest and reduced expression of myogenic regulatory factors. Many thousand of ?-gal+ nuclei are observed in MDSC-injected muscles while anyone is identified in myoblasts-injected ones : 55-64% are observed into muscle myofibers, 25-35% in satellite cell niche and ?10% in interstital tissue. Some of MDSC in satellite cell localization display Pax7 expression, revealing that they participate to maintain the satellite cell pool of dystrophic muscle. ?-gal+ nuclei presence is regularly associated with dystrophin expression. Also, ?-dystroglycan and sarcoglycans (??and????) are present throughout the sarcolemma of many hybrid fibers while utrophin is down-expressed. The outcome of our project provides novel insights into the myogenic potential of MDSC in clinically relevant context.
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