Satellite cells from equine soleus muscles were cultured and the expression of desmin, one of the markers of skeletal muscle cells, was examined. The presence ...
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Isolation of Satellite Cells from Equine Skeletal Muscle Chie SOETA1, Keitaro YAMANOUCHI 1, Telhisa HASEGAWA 2, Nobushige ISHIDA2, Harutaka MUKOYAMA2, Hideaki TOJO1* and Chikashi TACHI1 1
Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113 and 2Laboratory of Molecular and Cellular Biology, Equine Research Institute, Japan Racing Association, Utsunomiya 320, Japan
Satellite cells from equine soleus muscles were cultured and the expression of desmin, one of the markers of skeletal muscle cells, was examined. The presence of desmin was detected in some populations of mononucleated cells on day 3 of culture, indicating that equine satellite cells were successfully isolated. On day 5, desmin was localized at the edges of the elongated myotubes but when the equine myotubes matured, no particular localization of desmin expressed diffusely within the cells was seen. Although the significance of such changes in the localization of desmin within the differentiating equine skeletal muscle cells is unknown, these results suggested that desmin may be involved in the growth and development of skeletal muscle in this species. Key words: equine, satellite cells, desmin, culture
Skeletal muscle satellite cells are mononucleated cells that lie under or are embedded in the basal lamina of the myofibre [1]. They are resting during most of the adult life of animals, but these cells can be activated following injury [2]. Upon activation, they proliferate and finally differentiate and fuse with adjacent muscle fibre or with other satellite cells to form new multinucleated fibres [2]. Therefore, satellite cells play an indispensable role during skeletal muscle regeneration processes. In addition, these cells play a role in muscle hypertrophy by adding new nuclei to the preexisting fibres and this process is similar to the fusion of skeletal muscle cells seen during regeneration [2]. The horse is the only domestic animal that is exercised for the purpose of inducing muscle hypertrophy to improve performance. This is especially true of racing horses. Therefore, obtaining knowledge of the mechanism of postnatal muscular hypertrophy in this species will be of value in establishing an appropriate method for training horses. For this reason, in the present study, we have attempted to isolate equine skeletal muscle satellite cells and established an in vitro
This article was submitted March 12, 1998 and was accepted July 8, 1998. *corresponding author.
J. Equine Sci. Vol. 9, No. 3 pp. 97–100, 1998
culture system. The procedure used to isolate equine skeletal muscle satellite cells was according to previous reports [3–5] with some modifications by us. In brief, soleus muscles were taken from a stallion at the Equine Research Institute of the Japan Racing Association (Tokyo, Japan). The stallion was sacrificed as previously described [6]. The type of skeletal muscle as a source for harvesting satellite cells was chosen based on the preliminary experiments in which three kinds of skeletal muscles were used, i.e. soleus muscle, gluteus medius muscle and diaphragm. The soleus muscle was the tissue from which the highest yield of mononucleated cells was obtained (data not shown). The tissues were dissected free of connective and adipose tissues. Each muscle tissue was washed in Hank’s medium (Gibco BRL, NY), containing penicillin 100 U/ml, streptomycin 100 µg/ ml and fungizon 100 µg/ml. Connective tissue was further removed with scissors to minimize contamination of fibroblasts. The tissue was washed again in Hank’s medium containing penicillin 100 U/ ml, streptomycin 100 µg/ml, fungizon 100 µg/ml and 2% fetal bovine serum (FBS) (heat inactivated at 56˚C for 30 min) and cut into pieces. Pieces of the tissues were washed in phosphate buffered saline (PBS), containing penicillin 100 U/ml, streptomycin 50 µg/
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ml, fungizon 100 µg/ml and minced. The minced tissues were treated with 0.1% trypsin-0.02% EDTA in PBS at 37˚C for 1 hr with continuous stirring and tissue debris was removed by filtering through two layers of lens paper (Whatman, Maidstone, England). Cells were centrifuged at 1,200 rpm for 10 min at room temperature, resuspended in McCoy’s 5A medium (Gibco BRL, NY) containing penicillin 100 U/ml, streptomycin 50 µg/ml, gentamycin 50 µg/ml, 20% heatinactivated FBS, and plated at a density of 1 × 105 cells/ 10 ml/10 cm per culture dish and cultured at 37˚C in an atmosphere of 95% air: 5% CO 2 with saturated humidity. One to two hour(s) later, unattached cells containing skeletal muscle cells were re-plated on collagen-coated glass coverslips (three coverslips were placed in a 3.5 cm dish containing 2 ml of medium) at a density of 1 × 104 cells/2 ml/dish, and cultured for 10 days. The medium was changed every three days. Desmin, one of the intermediate filaments, has been shown to be a useful marker for identifying satellite cells in several species [7]. Therefore, an indirect immunofluorescent analysis for the expression of desmin was carried out as described previously [8] to identify equine satellite cells in the culture. The primary antibody used was anti-mouse desmin monoclonal antibody (Sigma, St. Louis MO). The cells grown on the glass coverslips were fixed in methanol for 20 min at − 20˚C and then incubated in 0.1% Triton X-100 in PBS for 15 min at room temperature. After washing in PBS, blocking solution (1% bovine serum albumin in PBS) was added. After overnight incubation at 4˚C, the blocking solution was discarded and the primary antibody (diluted to 1:50 with blocking solution) was applied. Incubation was performed for 1.5 hr at room temperature, followed by washing three times in blocking solution. Then the fluorescein-labeled secondary antibody (FITC labeled goat anti-mouse IgG1; Sigma, St Louis, MO; 1:100 dilution with blocking solution) was applied. After being incubated for 1 hr at room temperature, the specimens were washed in blocking solution and mounted with glycerol gel for microscopic observation. Preparations were observed under the microscope with an Olympus photo microscope equipped (B201; Olympus, Tokyo, Japan) with epi-illumination and specific filters for fluorescein. Microscopic observation under the phase contrast had not enabled us to distinguish the satellite cells from contaminated fibroblasts, due to the similarity in their morphological features (Fig. 1B). When the cells at 3 days of culture were stained for the presence of desmin, most of the desmin-positive cells were mononucleated
Fig. 1.
Desmin-positive mononuclear cells obtained from equine soleus muscle in vitro. Cells were isolated and cultured as described in the text. Immunocytochemical analysis was performed with anti-desmin antibody. At 3 days of culture, desmin-positive cells were mononucleated and some of them appeared to be elongating in shape (arrows) (A). B, phase contrast corresponding to (A). Scale bar, 35 µm.
and some of these cells appeared to be synchronously differentiating into elongated shapes (Fig. 1A). At 5 days of culture, the desmin-positive cells were mostly elongated, suggesting that these cells were undergoing fusion. It should be noted that, at this stage, the desmin within the cells was concentrated at the edge of the primary myotubes (Fig. 2A, C). Interestingly, the primary myotubes approached each other where localization of desmin was observed (Fig. 2C, D). This may represent the possibility that fusion of the cells would be taking place at the site where the desmin was localized. When the culturing period was extended for up to 7 days, mature myotubes were formed and no indication of fusion occurring was seen. At this period, desmin did not show further signs of particular localization and was expressed diffusely within the mature myotubes (Fig. 3A,
EQUINE SATELLITE CELLS
Fig. 2.
Localization of desmin in the primary equine myotubes formed in vitro. At 5 days of culture, the desmin positive cells were mostly the primary myotubes bearing several elongated nuclei (A and C). The desmin was localized at the edges of elongated cells (arrow in A). Note that the primary myotubes approached each other where desmin was concentrated (arrowheads in C). B and D, phase contrast corresponding to (A) and (C), respectively. Scale bar, 35 µm.
Fig. 3.
Diffused distribution of desmin in equine mature myotubes in vitro. At 7 days of culture, desmin-positive cells (arrows) were differentiated to mature myotubes. Note that at this stage of differentiation, desmin was localized diffusely within the cells (A). B, phase contrast corresponding to (A). Arrowheads, nuclei of the mature multinucleated myotube. Scale bar, 35 µm.
B). Although the biological significance of such changes in the localization of desmin is not yet known, we hypothesize that desmin is important in the process of differentiation of skeletal muscle cells. In this paper, we described the isolation and culture of equine skeletal muscle satellite cells. Establishment of an appropriate method for purifying equine satellite cells will be required to clarify the significance of the particular localization of desmin in primary myotubes.
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Acknowledgment This work was supported in part by grants-in-aid from the Equine Research Institute, Japan Racing Association, and the Ministry of Education, Science, Sports and Culture, Japan (No. 09760251), and Kanagawa Academy of Science and Technology Research (No. 9971032).
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