A population of cells resident within embryonic and newborn rat skeletal muscle is capable of differentiating into multiple mesodermal phenotypes

Abstract

We have previously shown a population of putative mesenchymal stem cells in the connective tissue surrounding embryonic avian skeletal muscle. These cells differentiate into at least five recognizable phenotypes in culture: fibroblasts, chondrocytes, myotubes, osteoblasts, and adipocytes. We have now isolated a similar population of cells from fetal and newborn rat skeletal muscle. Cells from rat leg muscle were dissected, minced, and then enzymatically digested with a collagenase-dispase solution. The dissociated cells were plated and allowed to differentiate into two recognizable populations: myotubes and stellate mononucleated cells. The cells were then trypsinized, filtered through a 20 microm filter to remove the myotubes, frozen at -80 degrees C, then thawed and replated. In culture the cells maintained their stellate structure. However, under treatment with dexamethasone, a nonspecific differentiating agent, seven morphologic conditions emerged: cells with refractile vesicles that stained with Sudan black B (adipocytes), multinucleated cells that 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), cells with 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-shaped cells that grew in a swirl pattern (fibroblasts). The initial population is tentatively classified as putative mesenchymal stem cells. The presence of these cells point to the existence of stem cells in the postembryonic mammal that could provide a basis for tissue regeneration as opposed to scar tissue formation during wound healing.

Clonogenic analysis reveals reserve stem cells in postnatal mammals: I. Pluripotent mesenchymal stem cells

“…Clonogenic analysis (Young et al, , 1998bRogers et al, 1995) has confirmed the existence of clonal populations of both categories of stem cells in prenatal avians and prenatal mice. Recent preliminary studies involving rabbits, rats, and humans (Pate et al, 1993;Lucas et al, 1995Lucas et al, , 1996Young et al, 1999), have suggested that the stem cells of postnatal animals may contain pluripotent stem cells in addition to the progenitor stem cells demonstrated in previous studies. The current study was designed to determine whether clonal populations of pluripotent stem cells exist in postnatal mammals.…”

“…Similar cultures treated with dexamethasone will exhibit the expression of multiple phenotypes. Thus, comparing the effects of treatment with dexamethasone and insulin can identify specific types of progenitor and pluripotent cells within an unknown population of cells (Young et al, 1992a(Young et al, ,b, 1993(Young et al, , 1998a(Young et al, ,b, 1999Young, 2000;Lucas et al, 1993Lucas et al, , 1995Pate et al, 1993;Rogers et al, 1995;Warejcka et al, 1996).…”

“…Mesodermal stem cells have been isolated and identified in multiple animal species, including avians (Young et al, 1992a, mice (Rogers et al, 1995;Saito et al, 1995;Young et al, 1998a), rats (Grigoriadis et al, 1988;Lucas et al, 1995Lucas et al, , 1996Dixon et al, 1996;, rabbits (Pate et al, 1993;Wakitani et al, 1994;Grande et al, 1995;R.G. Young et al, 1998), and humans (Caplan et al, 1997;Young et al, 1999;Young, 2000).…”

“…Preliminary studies involving postnatal rats, rabbits, and humans suggest that resident stem cells consist of both lineage-uncommitted pluripotent stem cells (Pate et al, 1993;Lucas et al, 1995Lucas et al, , 1996Young et al, 1999) as well as the established populations of lineage-committed progenitor stem cells (Mauro, 1961;Cruess, 1982;Campion, 1984;Owen, 1988;Beresford, 1989;Ailhaud et al, 1992;Grounds et al, 1992;Rickard et al, 1994;Vierck et al, 1996;Caplan et al, 1997;Prockop, 1997;Palis and Segel, 1998;McGuire, 1998;Ratajczak et al, 1998;Pittenger et al, 1999). The current clonogenic study was undertaken to determine whether clonal populations of pluripotent stem cells are present in the connective tissues of postnatal rats, and to examine their functional capabilities.…”

Clonogenic analysis reveals reserve stem cells in postnatal mammals. II. Pluripotent epiblastic‐like stem cells

“…The identity of specific types of progenitor and pluripotent cells within an unknown population of cells can be ascertained by comparing the effects of treatment with a progression factor and a general nonspecific lineage-induction agent (Young et al, 1992a(Young et al, , 1992b(Young et al, , 1998a(Young et al, , 1998b(Young et al, , 2001a(Young et al, , 2001bLucas et al, 1993Lucas et al, , 1995Pate et al, 1993;Rogers et al, 1995;Warejcka et al, 1996;Young, 2000Young and Black, 2004). Progression factors, such as insulin (at 2-5 g/ml), accelerate phenotypic expression in progenitor cells but have no effect on the induction of phenotypic expression in pluripotent stem cells.…”