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 1A Population of Cells Resident Within Embryonic and Newborn Rat Skeletal Muscle Is Capableof Differentiating Into Multiple Mesodermal PhenotypesPaul A. Lucas, Ph.D. Department of Surgery, Mercer University School of Medicine at theMedical Center of Central Georgia, Macon, GA 31201Andrew F. Calcutt, M.D. Department of Surgery, Mercer University School of Medicine at theMedical Center of Central Georgia, Macon, GA 31201Sheila S. Southerland, B.A. Department of Surgery, Mercer University School of Medicine at theMedical Center of Central Georgia, Macon, GA 31201J. Alan Wilson, M.D. Department of Surgery, Mercer University School of Medicine at theMedical Center of Central Georgia, Macon, GA 31201Richard L. Harvey, M.D. Department of Surgery, Mercer University School of Medicine at theMedical Center of Central Georgia, Macon, GA 31201Debra Warejcka, Ph.D. Department of Surgery, Mercer University School of Medicine at theMedical Center of Central Georgia, Macon, GA 31201Henry E. Young, Ph.D. Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA 31207Work should be attributed to: Department of Surgery, Mercer University School of Medicine atthe Medical Center of Central Georgia, Macon, GA 31201Correspondence and reprint requests should be addressed to: Paul A. Lucas, Ph.D. Departmentof Surgery, Hospital Box 140, Medical Center of Central Georgia, 777 Hemlock Street, Macon,GA 31201 Phone: 912-752-2093 Fax: 912-752-2047Running title: Mesenchymal stem cells in rat tissue
 
 2ABSTRACTWe have demonstrated a population of putative mesenchymal stem cells in the connective tissuesurrounding embryonic avian skeletal muscle. These cells differentiate into at least 5recognizable phenotypes in culture: fibroblasts, chondrocytes, myotubes, osteoblasts, andadipocytes. We have isolated a similar population of cells from fetal and newborn rat skeletalmuscle. Cells from rat leg muscle were dissected, minced, then enzymatically digested with acollagenase-dispase solution. The dissociated cells were plated and allowed to differentiate intotwo recognizable populations: myotubes and stellate mononucleated cells. The cells were thentrypsinized, filtered through a 20 µm filter to remove the myotubes, frozen at -80°C, then thawedand replated. In culture the cells maintained their stellate morphology. However, undertreatment with dexamethasone, a non-specific differentiating agent, 7 morphologies emerged:cells with refractile vesicles that stained with Sudan black B (adipocytes), multinucleated cellsthat spontaneously contracted in culture and stained with an antibody to myosin (myotubes),round cells whose extracellular matrix stained with Alcian blue, pH 1.0 (chondrocytes),polygonal cells whose extracellular matrix stained with Von Kossa's stain (osteoblasts), cellswith filaments that stained with an antibody to smooth muscle a-actin (smooth muscle cells),cells that incorporated acetylated-low density lipoprotein (endothelial cells), and spindle-shapedcells that grew in a swirl pattern (fibroblasts). The initial population is tentatively classified asputative mesenchymal stem cells. The presence of these cells point to the existence of stem cellsin the post-embryonic mammal that could provide a basis for tissue regeneration as opposed toscar tissue formation during wound healing.Key words: wound healing, mesenchyme, stem cells, dexamethasone
 
 3INTRODUCTION:Skeletal muscle has a limited capacity for repair in response to injury. Understanding thisrepair involves knowledge of both the cells involved and the local factors that influence the cells.Myosatellite cells are mononucleated, quiescent, myogenically committed stem cells locatedbetween the muscle sarcolemma and the overlying basement membrane (1,2). These cells arethought to be involved in muscle maintenance and provide the stem cells for muscle repair.Following focal injury to muscle fibers, these cells proliferate and migrate, within limits, to thesite of injury and fuse to form new myotubes. Muscle regeneration studies involving musclemincing, muscle grafting, and focal injury to the myofibers by temperature extremes, ischemia,anesthetics, and snake venom have all noted formation of regenerating myofibers within theconnective tissue scar immediately adjacent to intact myofibers (3-8).However, a localized migration of satellite cells does not explain the results of otherstudies demonstrating the formation of myofibers embedded within the connective tissue scar atsome distance from intact myofibers (9,10). Satellite cells also do not explain the numerousstudies showing the de novo induction, using demineralized bone matrix or proteins derived fromit, of cartilage and bone in muscle tissue (11-14). Indeed, minced muscle explants grown ondemineralized bone matrix or with media containing soluble bone proteins demonstrate thedifferentiation of cartilage (15,16). These studies imply the existence of a more primitive stemcell located within skeletal muscle that is capable of differentiating into skeletal muscle,cartilage, and bone.A population of cells from day 11 embryonic chick leg muscle capable of differentiatinginto cartilage, skeletal muscle, bone, fat, and connective tissue has been reported (17). Thecurrent study was designed to determine the existence of a similar population of cells in ratskeletal muscle.
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