Transient neonatal denervation alters the proliferative capacity of myosatellite cells in dystrophic (129ReJ^dy/dy) muscle

Abstract

It has been previously shown that transiently denervated, neonatal dystrophic muscle fails to undergo the degeneration-regeneration cycle characteristic of murine dystrophy (Moschella and Ontell, 1987). Thus, the myosatellite cells (myogenic stem cells) in these muscles have been spared the mitotic challenge to which dystrophic myosatellite cells are normally subjected early in the time course of the disease. By in vitro evaluation of the proliferative capacity of myosatellite cells derived from extensor digitorum longus (EDL) muscles of 100-day-old genetically normal (+/+) and genetically dystrophic [dy/dy (129ReJdy/dy)] mice and from muscles of age-matched mice that had been neonatally denervated (by sciaticotomy) and allowed to reinnervate, it has been possible to directly determine whether the cessation of spontaneous regeneration in older dy/dy muscles in vivo, is due to an innate defect in the proliferative capacity of the myosatellite cells or exhaustion of the myosatellite cells' mitotic activity during the regenerative phase of the disease. This study demonstrates that transient neonatal denervation of dystrophic muscle (Den.dy/dy) increases the number of muscle colony-forming cells (MCFs) per milligram of wet weight muscle tissue, increases the plating efficiency, and significantly increases the in vitro mitotic activity of dystrophic myosatellite cells toward normal values. The increased mitotic capability of myosatellite cells derived from Den.dy/dy muscle as compared to unoperated dy/dy muscle suggests that there is no innate defect in the proliferative capacity of the myosatellite cells of dy/dy muscles and that the cessation of spontaneous regeneration in the dy/dy muscles is related to the exhaustion of their myosatellite cells' mitotic capability.

Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD.

“…Despite expression of MyoD and ␤ -galactosidase in the explanted fibroblasts, no muscle differentiation took place in vitro. Next, skeletal myoblasts were isolated from the limbs of inbred Fischer rat pups (35) and transplanted into healing freeze-thaw lesions in adult Fischer rats. The engrafted myoblasts fused to form multinucleated myotubes and expressed skeletal MHC isoforms.…”

Effect of Injecting Primary Myoblasts Versus Putative Muscle-Derived Stem Cells on Mass and Force Generation inmdxMice

“…Anesthetized mice were killed by cervical dislocation, and muscles from both legs of the mice were pooled and minced. Isolation of myosatellite cells and culture conditions were modified from Ontell et al (1992). Minced muscles were incubated at room temperature for 10 min in 0.025% collagenase (type 1; Worthington, Freehold, NJ) in Ca 21 -and Mg 21 -free Puck's saline G (Bonner and Hauschka, 1974) and centrifuged (900 rpm for 8 min).…”

Limitations of nlsβ-galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer

“…Cells of the myogenic cell line MM14 (Clegg et al, 1987; provided by Stephen Hauschka, University of Washington, Seattle) were cultured in proliferation media (Ham's F10 medium; Sigma, St. Louis, MO) supplemented with 15% horse serum (preselected for favoring myoblast division; Atlanta Biologicals, Norcross, GA) and 2 ng/ml of human recombinant fibroblast growth factor (b-FGF; provided by Stephen Hauschka, University of Washington, Seattle), as previously described (Ontell et al, 1992). Stable transfection was achieved as follows: 8 µg of RV3FnlslacZ3 DNA construct and 80 µg of lipofectamine (GibcoBRL) premixed in 600 µl of serum free Ham's F10 medium were added to a 60-mm culture dish containing 2 3 10 5 MM14 cells in 2.4 ml of Ham's F10 medium supplemented with 5% horse serum and 2 ng/ml of b-FGF.…”

“…The cells were continuously selected and passaged (cf. Ontell et al, 1992) for 8 weeks. Thereafter, some plates were seeded at low density and fed with medium favoring differentiation (Ham's F10, 5% horse serum, and 1.9 mg/ml insulin), and others were seeded at clonal density and fed with proliferation medium.…”