Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages

Abstract

Adult skeletal muscle contains populations of satellite cells and muscle-derived stem cells that are capable of forming multinucleate myotubes. The purpose of this study was to determine the phenotype of cells isolated from a common satellite cell isolation and passaging procedure from whole skeletal muscle. To ascertain the characteristics of the cellular phenotype, the myogenic markers MyoD and desmin, the satellite-cell-specific marker Pax7, and the haemopoietic stem cell markers CD34 and CD45 were examined by immunohistochemical analysis. Immediately after isolation, > 90% myogenic marker-positive cells were positive for desmin, MyoD and Pax7. In contrast, approximately 10% of the isolated cells expressed only CD34 or CD45. After three passages, the percentage of cells that were positive for the myogenic markers desmin, MyoD and Pax7 was reduced to approximately 55%, while the population of CD34- or CD45-positive cells increased to approximately 30% after the third passage. Immunohistochemical detection of bromodeoxyuridine demonstrated that the number of proliferating cells decreased progressively after each passaging. Finally, after the third passage the percentage of nuclei in myotubes decreased from 46.7% to 12.5%. Since passaging of muscle progenitor cells is common practice, the results of the current report suggest that characterization of cell heterogeneity needs to be made frequently.

Figure 1

Immunohistochemical analyses with markers for myogenic, satellite cell and haemopoietic stem cells in primary muscle progenitor cells isolated from adult rats at the time of isolation. Desmin (a,a′) and MyoD (b,b′) were used as myogenic markers, Pax7 (c,c′) is shown as a satellite cell marker, CD34 (d) and CD45 (e) were used as haemopoietic stem cell markers. Desmin is expressed in the cytoplasm of isolated bipolar cells (a′, arrowheads) and the smaller, circular, non‐bipolar cells (a′, arrows). MyoD and Pax7 are present in the nuclei of the bipolar cells (b′,c′, arrowheads) and the smaller, circular, non‐bipolar cells (b′,c′, arrows). CD34 or CD45 are found in the smaller, circular, non‐bipolar cells (d,e, arrows), but not in the isolated bipolar cells. Myotubes were never observed, verifying that these markers were associated with muscle precursor cells and not with differentiating cells. Bars indicate 500 µm.

Figure 2

The expression profiles of myogenic, satellite cell, and haemopoietic stem cell markers during passaging of rat primary muscle progenitor cells. A total of at least 100 cells was counted in each sample. The data are means ± SE. The SE was calculated from three independent experiments performed on separate sets of animals. The changes in the percentage of cells expressing desmin, MyoD, Pax7, CD34 and CD45 between unpassaged cells (#0 on the x‐axis) and the third passage group (#3 on the x‐axis) were significantly different (*P < 0.05).

Figure 3

Effect of passaging on rat primary muscle progenitor cell proliferation. Muscle progenitor cell activation was assayed by BrdUrd incorporation in the adult rat muscle precursor cell culture. Cells were pulse‐labelled for 1 h with 10 µm BrdUrd before staining. The percentage of BrdUrd‐labelled cells was used as an indicator of activation and proliferating cells. Error bars represent the SE from three independent experiments performed on separate sets of animals. A total of at least 100 cells was counted in each sample. Significantly (P < 0.01) different from **unpassaged cells (#0 on the x‐axis), ^††one passage (#1 on the x‐axis), and ^‡‡two passage (#2 on the x‐axis) groups.

Figure 4

Differentiation potential of isolated progenitor cells decreases during passaging. Representative photograph of myotubes derived from unpassaged cells (a) and cells passaged three times (b) stained with a monoclonal antibody specific for sarcomeric myosin (MF20). The myotubes derived from unpassaged cells were large and contained numerous nuclei (a). In contrast, myotubes derived from three‐passaged cells were small with fewer incorporated nuclei (b).