DEVELOPMENTAL
BIOLOGY
55, 402-407
(1977)
In Vitro Myogenesis of the Mononucleate Cells Derived Regenerating Muscles of Adult Mice T. Laboratory
of Cell Biology,
KAGAWA,
E.
CHIKATA,
Toneyama
National
Hospital,
August
2,1976;
accepted
Received
AND 5-l-1,
J.
TANI
Toneyama-cho,
in revised
form
from
October
Toyonaka-shi,
Osaka,
Japan
7, 1976
Regeneration was induced in the matured skeletal muscles of adult mice by intramuscular injection of hypertonic saline. After passing through the degeneration phase, the regeneration of muscle took place and proceeded with considerable synchrony. Regenerating myogenic cells were isolated from the saline-treated muscles by cold trypsinization with the aid of Yaffe’s selective plating procedure [Yaffe, D. (1968) Proc. Nat. Acad. Sci. USA 61, 477-4831. The myogenic cells thus isolated readily developed into muscle fibers possessing cross striations and the ability to contract when cultured in vitro. INTRODUCTION
MATERIALS
The skeletal muscles of homeothermal animals are capable of regeneration when they are injured by pathological, chemical, and physical agents (7-9, 15, 16, 19, 20). Pogogeff and Murray (17) demonstrated that mature skeletal muscles of adult humans and rats were potent enough to regenerate in tissue culture and that mononucleate spindle cells migrated out from muscle explants. In muscle regeneration, myotubes were formed by the fusion of mononucleate spindle cells (14), as in myogenesis in embryos (5). The references cited above suggest the possibility that myogenic cells participating in muscle regeneration may be obtained from the regenerating skeletal muscles of adult homeothermal animals. This study was designed to isolate and cultivate mononucleate myogenic cells from mature skeletal muscles of adult mice in which regeneration bad been experimentally induced. As a means to induce muscle regeneration, we used the intramuscular injection of hypertonic saline. This procedure was found to be quite simple and satisfactory for providing regenerating muscles as a source of myogenic cells capable of differentiation when cultured in vitro. 0 1977 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Adult C57B1/6J mice (older than 8 weeks) were used throughout. One-tenth milliliter of 20% NaCl, dissolved in distilled water, was injected into femoral muscles. The lesions formed were recovered and used as histological preparations at 2 hr and 1,2,3, 4,5, 8, and 11 days after injection. The recovered specimens were fixed in Bouin’s solution, sectioned at 5 pm and stained with Mallory’s triple staining or Mayer’s hematoxylin-eosin. For cell culture, anterior femoral muscles were removed at 1, 2, 3, 4, 5, and 7 days after saline injections. The recovered tissues were cut into small fragments with a pair of knives. The minced muscles (wet weight of a leg muscle exposed to tryptic digestion averaged 100 mg) were washed twice with calcium-magnesium-free saline buffered with phosphate (PBS) and digested with 0.125% trypsin (dissolved in PBS) at 4°C for 3 hr with stirring. The resulting cell suspension, which consisted of dissociated single cells, was transferred into a centrifuge tube containing an equal volume of “the fresh medium” to stop digestion. “The fresh medium” consisted of 85% Eagle’s minimum essential medium (MEM), 10% horse serum and 5% chick 402
Copyright All rights
AND
403
BRIEF NOTES
embryo extract (1:l). Tryptic digestion was continued for an additional 3 hr on the tissues still remaining as small fragments. Then both cell suspensions were pooled together, and filtered through a platinum mesh (150 mesh) and two layers of lens paper set in a Millipore filter holder. After centrifugation at 800 rpm for 10 min, the cell pellet was resuspended in “the growth medium,” which consisted of 90% conditioned medium and 10% fetal calf serum. The number of fibroblasts mixed in the cell population was effectively reduced by the selective plating procedure given by Yaffe (21); 3 x 10” cells were seeded in a final volume of 5 ml of “the growth medium” and incubated for 30 min at 37°C. By 30 min most myogenic cells were found to be floating in the medium, whereas the majority of the fibroblasts was already attached to the bottom of a culture dish. The final cultures were established by plating 2 x lo” cells in 2 ml of “the growth medium” into 30-mm Nunclon plastic petri dishes (Copenhagen, Denmark) coated with 0.1% gelatin (18). Incubation was continued at 37°C in a humidified atmosphere gassed with a mixture of 5% CO, and 95% air. The cultures were fed at 1 and 4 days of incubation. The conditioned medium was prepared as follows: Five milliliters of “the fresh medium” was exposed to primary or secondary confluent cultures originated from regenerating mouse skeletal muscles in 50-mm Nunclon petri dishes. After 24 hr, the medium was collected, centrifuged at 4000 rpm for 20 min to exclude viable cells and debris and then stored at 4°C until use. Chick embryo extract was prepared from whole II-day embryos of White Leghorn. Cell type analysis of the cell population isolated by the present procedures was done morphologically on phase contrast microphotographs of the cultures (24 hr old). Some cultures of the same age stained with Giemsa were also observed under a light microscope. Myogenic cells and fibroblasts were defined according to
the morphological criteria given by Konigsberg (12). RESULTS
Histological observation on muscle regeneration induced by saline injection. From 2 to 24 hr after the injection of hypertonic saline into the femoral region, degenerative changes, varying from a slight disorganization of myofibrillar structures to hyaline degeneration of the sarcoplasma, were observed in muscle fibers in the region surrounding the site of the injection (Fig. 2). Sarcolemmal nuclei of necrotic fibers were pycnotic at 2 hr and a small number of spindle cells was observed around the injured fibers at 24 hr after the injury. At 2 hr after saline injection, many inflammatory cells, consisting predominantly of polymorphonuclear cells, were observed near the ruptured capillaries in the spaces between injured fibers. In the 24-hr lesion, many inflammatory cells assembled around necrotic fibers and some of them infiltrated into necrotic fibers. In the 2-day lesion, many spindle cells were observed along the sarcolemma of injured fibers and sometimes formed so-called “buddings” at the tips of the fragmented fibers. The exact origin of the spindle cells, whether from buddings (20) or from satellite cells (131, has not been determined since the present observations were limited only to a light microscopy. The nuclei of spindle cells, which contained one or two nucleoli, were vesicular in shape and some 15
FIG. 1. Changes in cell yield per leg during muscle regeneration. Four to ten legs were used in each experiment (see text). Cell yield before (-0-j and after (-3-j selection.
404
DEVELOPMENTAL
BIOLOGY
FIGURES
VOLUME
2-9
55, 1977
BRIEF NOTES
showed mitotic figures. In the 3-day lesion, spindle cells increased in number and were arranged along the sarcolemma of necrotic fibers (Fig. 3). Few, if any, multinucleate myotubes were observed. In the 4-day lesion, the number of spindle cells definitely decreased, whereas multinucleate myotubes increased in number and grew in length along the sarcolemma of necrotic fibers (Fig. 4). These findings suggested that the regenerative changes proceeded with considerable synchrony. Myotubes had large, vesicular nuclei arranged in one or two rows in sarcoplasma. No mitotic figures were detected among them. Some myotubes were connected with the ends of muscle fibers which survived without being injured by the saline treatment. Longitudinal myofibrillar structures had been formed by 4 days and cross striations were observed by 5 days after the injection. As muscle regeneration proceeded, the number of inflammatory cells decreased. From 5 to 11 days after the injury, the regenerative changes advanced further. The 11-day lesion consisted of two areas; the central necrotic area and the regenerative area surrounding the central necrotic area. Only a small number of degenerated fibers were left in the central necrotic area, surrounded by granular, homogeneous ground substances and connective tissues. In the regenerative area, many long striated muscle fibers were reconstructed, orienting themselves parallel to the muscle fibers that had survived the
405
saline injection without injury. The reconstrutted muscle fibers in the regenerative area could be easily distinguished from the matured muscle fibers not injured by the saline treatment, since the large, vesicular nuclei of the former were found isolated or in rows in the center of the sarcoplasma (Fig. 5). As controls, 0.1 ml of 0.9% NaCl solution was injected into femoral muscles resulting in degeneration with a subsequent regeneration, but the regenerative tissues produced were too small to provide enough myogenic cells to construct a useful cell culture system of myogenesis. Myogenesis in cell culture. An attempt was made to examine the differentiating capacity in vitro of the mononucleate cells isolated from the mature skeletal muscles in which regeneration had been induced. The cell population prepared from leg muscles consisted of almost 100% single mononucleate cells (Fig. 6). Cell viability was found to be more than 80%). The cell yield per leg increased for 3 days after the saline treatment and then declined abruptly (Fig. 1). It was about 15 x 10: at 3 days after the injection and about half of them were removed using the selective procedure described above. The ratio of the number of cells attached to culture dishes to the number of cells inoculated ranged from 65 to 85% when the inoculum sizes were between IO” and 8 x IO” per plate. All cell populations, obtained at various times after the saline injection, invariably
FIG. 2. Twenty-four-hour lesion. Muscle fibers surrounding the site of the hypertonic saline InJection show fragmentation and degeneration. x 190. FIG. 3. Three-day lesion. Mononucleate spindle cells are observed to be arranged along sarcolemma of necrotic fibers. Mitotic figures are seen among spindle cells, x 580. FIG. 4. Four-day lesion. Note that multinucleate myotubes occupy the regenerating area of a muscle. No mitotic figures are seen among the nuclei of myotubes. Compare with Fig. 3. x 190. FIG. 5. Eleven-day lesion. Vesicular sarcolemmal nuclei placed centrally either isolated or in rows show that these fibers are newly formed, Cross striations are seen in fibers. x 190. FIG. 6. Mononucleate single cells liberated from regenerating skeletal muscles (3 days after the jn.jectionj. After selection. Phase contrast. x 270. FIG. 7. Twenty-four hours after plating. The culture consists of mononuleate myogentc cells, fibroblasts, macrophages, and leukocytes. Phase contrast, x 270. FIG. 8. Ten-day-old culture. Muscle fibers developed. Fixed with ethanol, Phase contrast. x 210. FIG. 9. Ten-day-old culture. Note cross striations. Fixed with ethanol. Phase contrast. x 940.
406
DEVELOPMENTAL
BIOLOGY
formed muscle fibers in vitro and no differences were detected concerning their myogenic processes and morphology under the present culture conditions. For this reason, the description will be limited to the differentiating processes, in vitro, of the cells obtained from the leg muscles at 3 days after hypertonic saline injection. When plated, viable cells settled down on culture dishes and began to elongate within 12 hr. At 24 hr of incubation, the cultures consisted of mononucleate, myogenic cells (450/o), fibroblasts (7%), and macrophages and leukocytes (48%) (Fig. 7). Therefore, starting from the muscles of a leg in which regeneration was experimentally induced, approximately 2 x lo” myogenic cells were isolated and successfully cultured under the present conditions. Myogenic cells and fibroblasts started to propagate within 48 hr after plating, whereas macrophages and leukocytes were overgrown by them during the first 3 days of culture; consequently, the cultures consisted of mostly myogenic cells and fibroblasts at 3 days of culture. The ratio of the number of myogenic cells to that of fibroblasts was found to be 1O:l to 10:1.5 from 1 day through 5 days of culture. After 2 days of culture, myogenic cells became gradually smaller in size and rounded and began to assemble, resulting in an oriented pallisade. Among them an intense cell fusion occurred, forming multinucleate myotubes, which increased in number and grew in length during the next 3 days of culture. By 5 days after most mononucleate myogenic plating, cells had been incorporated into multinucleate myotubes. In the center of the myotubes, one or two rows of large, vesicular nuclei possessing one or two nucleoli were observed. With the advancement of myogenesis, the myotubes became longer and thinner. Cross striations were observed in some fibers at 6 days and in all fibers at 10 days of incubation (Figs. 8 and 9). Muscle fibers with cross striations in the present cultures showed only occasional, sponta-
VOLUME
55, 1977
neous contractions. It was found, however, that the use of a culture medium consisting of 89% MEM, 10% horse serum, and 1% chick embryo extract greatly enhanced the muscle fibers’ ability to contract. When transferred to the above culture medium after 4 days of culture, virtually all muscle fibers showed spontaneous contraction at 10 days of culture. An attempt at cell culture was extended to the femoral muscles of adult mice that had not received saline injection. Approximately 5 x lo4 cells per leg were obtained by the same procedure used for the salinetreated muscles. During the process of selective plating, cell number decreased drastically to about one-fortieth of the original cell number. The cells obtained after the selective plating procedure formed muscle fibers under the culture conditions described above. DISCUSSION
Histological findings revealed that the regenerative changes induced by the intramuscular injection of hypertonic saline were similar to those caused by other chemical and physical agents (4). Available data showed that marcain induced synchronous regeneration in the mature skeletal muscles of rats (11). Three features in the present system, however, seem worth mentioning: (a) Hypertonic saline may ultimately become a physiological substance for muscles when it is diluted in tissues; (b) saline injection induced muscular regeneration on a scale large enough to give many myogenic cells without any failure; and (cl histological observation demonstrated that muscle regeneration in the present system proceeded with considerable synchrony. These highly advantageous features for analyzing muscle regeneration have seldom been met by the previously reported experimental means of inducing muscle regeneration in vivo. The injected hypertonic saline may diffuse rapidly into the surrounding tissues, providing a concentration appropriate for the in-
BRIEF
duction of muscle regeneration in certain areas of the tissues and causing an instant injury in the tissues without showing any lag effect. These points may be quite important in attaining the conditions necessary for the synchronous advancement of regenerating processes. In summary, the intramuscular injection of hypertonic saline seems a very simple, yet quite reliable, method to induce regeneration in the skeletal muscles of adult mice, especially for the purpose of obtaining a large enough quantity of cells from the regenerating tissues for the purpose of cell culture studies. The present result showed that approximately 2 x lo” myogenic cells with the potency to differentiate into muscle fibers in vitro were isolated from muscles of a leg of an adult mouse in which regeneration was experimentally induced. Numerous data on the myoblasts from embryos have been accumulated since the pioneering work by Konigsberg (12), but only limited information was available concerning the myogenic cells participating in muscle regeneration in adults. In this sense, the studies on the role of satellite cells in muscle regeneration, in vitro, of a juvenile quail (13) and on the myoblasts isolated from a regenerating tail of a lizard (6) are very important. Mononucleate myogenic cells have been obtained from the explant cultures of skeletal muscles of adult humans (1, 3, 10) and directly from matured skeletal muscles of rats (2). To construct a useful culture system of myogenesis from adult muscles, such features as a large cell yield, the purity of cell population, and the uniformity of cell age are thought to be indispensable prerequisites. The myogenie cell population isolated by the present method may serve as a useful culture system for the analysis of the regenerating
407
NOTES
and differentiating muscles.
phenomena
in skeletal
We thank Dr. Yoshihiro Kato, Mitsubishi-Kasei Institute of Life Sciences, for his helpful advice and critical reading of the manuscript. The authors are also grateful to Dr. Kengo Yamada. Tokushima University, for his encouragement throughout this study. This study was partly supported by a research grant for muscular dystrophy from the Ministry 01 Health and Welfare of Japan. REFERENCES 1. BATESON, R. G.. HINDLE, D., and WARREN, J. (1972). J. Neural. Sci. 15, 183-191. R. (19751. Anat. Rec. 180. 645-662. 2. BISHOFF, 3. BISHOP, A., GALLUP, B., SKEATE, Y., and Dr.BOWITZ, Y. (1971). J. Neural. Sci. 13, 333-350. 4. BRUCE, M. C. (1974). Amer. J. Anat. 137. 119150. 5. CAPER, C. R. (1960). J. Biophys. Biochem. 7, 559-579. 6. Cox, P. G. (1968). J. Morphol. 126, 1-18. 7 DENNY-BROWN, D. (19511. J. NPuropathol. Exp Neural. 10, 94-96. 8 FISCHBACH, D. K., and FISCHBACH, H. R. (19321 Amer. J. Path. 8, 211-217. 9 FORBUS, W. D. (1926). Arch. Pathol. 2, 486-499. 10. GALLUP, B., STRUGALSKA-CYNOWSKA. H., and DUBOWITZ, V. (1972). J. Neural. Sci. 17. 109.. 125.
11. HALL-CRAGGS, 349-358. 12. KONIGSBERG, 13. KONIGSBERG, 14. 15. 16. 17. 18. 19.
20. 21.
E. C. B. (1974).
Exp.
Neuroi.
13.
1. R. (1963) Science 140, 1273-1284. U. R., LIPTON. B. H., and KONIGSBERG, I. R. (1975). Deuelop. Biol. 1.5, 260-275. LASH, J. W., HOLTZER, H., and SWIFT, H. (1957 Anat. Rec. 128. 679-697. LE GROS CLARK, W. E. (1946). J. Anat. X0.24-36, MILLER, W. G. (1934). J. Pathol. Ract. 38. 14% 151. POGOGEFF, I. A.. and MURRAY, M. R. 119461. Anat. Rec. 95, 321-335. RICHLER, C., and YAFFE, D. (1970). Dwe/oq. Biol. 23, l-22. WALDEYER, W. (1865). Arch. Pathol. Anat. Phyiol. 33, 473-514, cited from (20). WALTON, cl. N., and ADAMS, R. D. (1956). J. Pathol. Bact. 72, 273-298. YAFFE, D. (19681. Proc. Nat. Acad. SCL. 1iSA 61, 477-483.
BIOLOGY
55, 402-407
(1977)
In Vitro Myogenesis of the Mononucleate Cells Derived Regenerating Muscles of Adult Mice T. Laboratory
of Cell Biology,
KAGAWA,
E.
CHIKATA,
Toneyama
National
Hospital,
August
2,1976;
accepted
Received
AND 5-l-1,
J.
TANI
Toneyama-cho,
in revised
form
from
October
Toyonaka-shi,
Osaka,
Japan
7, 1976
Regeneration was induced in the matured skeletal muscles of adult mice by intramuscular injection of hypertonic saline. After passing through the degeneration phase, the regeneration of muscle took place and proceeded with considerable synchrony. Regenerating myogenic cells were isolated from the saline-treated muscles by cold trypsinization with the aid of Yaffe’s selective plating procedure [Yaffe, D. (1968) Proc. Nat. Acad. Sci. USA 61, 477-4831. The myogenic cells thus isolated readily developed into muscle fibers possessing cross striations and the ability to contract when cultured in vitro. INTRODUCTION
MATERIALS
The skeletal muscles of homeothermal animals are capable of regeneration when they are injured by pathological, chemical, and physical agents (7-9, 15, 16, 19, 20). Pogogeff and Murray (17) demonstrated that mature skeletal muscles of adult humans and rats were potent enough to regenerate in tissue culture and that mononucleate spindle cells migrated out from muscle explants. In muscle regeneration, myotubes were formed by the fusion of mononucleate spindle cells (14), as in myogenesis in embryos (5). The references cited above suggest the possibility that myogenic cells participating in muscle regeneration may be obtained from the regenerating skeletal muscles of adult homeothermal animals. This study was designed to isolate and cultivate mononucleate myogenic cells from mature skeletal muscles of adult mice in which regeneration bad been experimentally induced. As a means to induce muscle regeneration, we used the intramuscular injection of hypertonic saline. This procedure was found to be quite simple and satisfactory for providing regenerating muscles as a source of myogenic cells capable of differentiation when cultured in vitro. 0 1977 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Adult C57B1/6J mice (older than 8 weeks) were used throughout. One-tenth milliliter of 20% NaCl, dissolved in distilled water, was injected into femoral muscles. The lesions formed were recovered and used as histological preparations at 2 hr and 1,2,3, 4,5, 8, and 11 days after injection. The recovered specimens were fixed in Bouin’s solution, sectioned at 5 pm and stained with Mallory’s triple staining or Mayer’s hematoxylin-eosin. For cell culture, anterior femoral muscles were removed at 1, 2, 3, 4, 5, and 7 days after saline injections. The recovered tissues were cut into small fragments with a pair of knives. The minced muscles (wet weight of a leg muscle exposed to tryptic digestion averaged 100 mg) were washed twice with calcium-magnesium-free saline buffered with phosphate (PBS) and digested with 0.125% trypsin (dissolved in PBS) at 4°C for 3 hr with stirring. The resulting cell suspension, which consisted of dissociated single cells, was transferred into a centrifuge tube containing an equal volume of “the fresh medium” to stop digestion. “The fresh medium” consisted of 85% Eagle’s minimum essential medium (MEM), 10% horse serum and 5% chick 402
Copyright All rights
AND
403
BRIEF NOTES
embryo extract (1:l). Tryptic digestion was continued for an additional 3 hr on the tissues still remaining as small fragments. Then both cell suspensions were pooled together, and filtered through a platinum mesh (150 mesh) and two layers of lens paper set in a Millipore filter holder. After centrifugation at 800 rpm for 10 min, the cell pellet was resuspended in “the growth medium,” which consisted of 90% conditioned medium and 10% fetal calf serum. The number of fibroblasts mixed in the cell population was effectively reduced by the selective plating procedure given by Yaffe (21); 3 x 10” cells were seeded in a final volume of 5 ml of “the growth medium” and incubated for 30 min at 37°C. By 30 min most myogenic cells were found to be floating in the medium, whereas the majority of the fibroblasts was already attached to the bottom of a culture dish. The final cultures were established by plating 2 x lo” cells in 2 ml of “the growth medium” into 30-mm Nunclon plastic petri dishes (Copenhagen, Denmark) coated with 0.1% gelatin (18). Incubation was continued at 37°C in a humidified atmosphere gassed with a mixture of 5% CO, and 95% air. The cultures were fed at 1 and 4 days of incubation. The conditioned medium was prepared as follows: Five milliliters of “the fresh medium” was exposed to primary or secondary confluent cultures originated from regenerating mouse skeletal muscles in 50-mm Nunclon petri dishes. After 24 hr, the medium was collected, centrifuged at 4000 rpm for 20 min to exclude viable cells and debris and then stored at 4°C until use. Chick embryo extract was prepared from whole II-day embryos of White Leghorn. Cell type analysis of the cell population isolated by the present procedures was done morphologically on phase contrast microphotographs of the cultures (24 hr old). Some cultures of the same age stained with Giemsa were also observed under a light microscope. Myogenic cells and fibroblasts were defined according to
the morphological criteria given by Konigsberg (12). RESULTS
Histological observation on muscle regeneration induced by saline injection. From 2 to 24 hr after the injection of hypertonic saline into the femoral region, degenerative changes, varying from a slight disorganization of myofibrillar structures to hyaline degeneration of the sarcoplasma, were observed in muscle fibers in the region surrounding the site of the injection (Fig. 2). Sarcolemmal nuclei of necrotic fibers were pycnotic at 2 hr and a small number of spindle cells was observed around the injured fibers at 24 hr after the injury. At 2 hr after saline injection, many inflammatory cells, consisting predominantly of polymorphonuclear cells, were observed near the ruptured capillaries in the spaces between injured fibers. In the 24-hr lesion, many inflammatory cells assembled around necrotic fibers and some of them infiltrated into necrotic fibers. In the 2-day lesion, many spindle cells were observed along the sarcolemma of injured fibers and sometimes formed so-called “buddings” at the tips of the fragmented fibers. The exact origin of the spindle cells, whether from buddings (20) or from satellite cells (131, has not been determined since the present observations were limited only to a light microscopy. The nuclei of spindle cells, which contained one or two nucleoli, were vesicular in shape and some 15
FIG. 1. Changes in cell yield per leg during muscle regeneration. Four to ten legs were used in each experiment (see text). Cell yield before (-0-j and after (-3-j selection.
404
DEVELOPMENTAL
BIOLOGY
FIGURES
VOLUME
2-9
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BRIEF NOTES
showed mitotic figures. In the 3-day lesion, spindle cells increased in number and were arranged along the sarcolemma of necrotic fibers (Fig. 3). Few, if any, multinucleate myotubes were observed. In the 4-day lesion, the number of spindle cells definitely decreased, whereas multinucleate myotubes increased in number and grew in length along the sarcolemma of necrotic fibers (Fig. 4). These findings suggested that the regenerative changes proceeded with considerable synchrony. Myotubes had large, vesicular nuclei arranged in one or two rows in sarcoplasma. No mitotic figures were detected among them. Some myotubes were connected with the ends of muscle fibers which survived without being injured by the saline treatment. Longitudinal myofibrillar structures had been formed by 4 days and cross striations were observed by 5 days after the injection. As muscle regeneration proceeded, the number of inflammatory cells decreased. From 5 to 11 days after the injury, the regenerative changes advanced further. The 11-day lesion consisted of two areas; the central necrotic area and the regenerative area surrounding the central necrotic area. Only a small number of degenerated fibers were left in the central necrotic area, surrounded by granular, homogeneous ground substances and connective tissues. In the regenerative area, many long striated muscle fibers were reconstructed, orienting themselves parallel to the muscle fibers that had survived the
405
saline injection without injury. The reconstrutted muscle fibers in the regenerative area could be easily distinguished from the matured muscle fibers not injured by the saline treatment, since the large, vesicular nuclei of the former were found isolated or in rows in the center of the sarcoplasma (Fig. 5). As controls, 0.1 ml of 0.9% NaCl solution was injected into femoral muscles resulting in degeneration with a subsequent regeneration, but the regenerative tissues produced were too small to provide enough myogenic cells to construct a useful cell culture system of myogenesis. Myogenesis in cell culture. An attempt was made to examine the differentiating capacity in vitro of the mononucleate cells isolated from the mature skeletal muscles in which regeneration had been induced. The cell population prepared from leg muscles consisted of almost 100% single mononucleate cells (Fig. 6). Cell viability was found to be more than 80%). The cell yield per leg increased for 3 days after the saline treatment and then declined abruptly (Fig. 1). It was about 15 x 10: at 3 days after the injection and about half of them were removed using the selective procedure described above. The ratio of the number of cells attached to culture dishes to the number of cells inoculated ranged from 65 to 85% when the inoculum sizes were between IO” and 8 x IO” per plate. All cell populations, obtained at various times after the saline injection, invariably
FIG. 2. Twenty-four-hour lesion. Muscle fibers surrounding the site of the hypertonic saline InJection show fragmentation and degeneration. x 190. FIG. 3. Three-day lesion. Mononucleate spindle cells are observed to be arranged along sarcolemma of necrotic fibers. Mitotic figures are seen among spindle cells, x 580. FIG. 4. Four-day lesion. Note that multinucleate myotubes occupy the regenerating area of a muscle. No mitotic figures are seen among the nuclei of myotubes. Compare with Fig. 3. x 190. FIG. 5. Eleven-day lesion. Vesicular sarcolemmal nuclei placed centrally either isolated or in rows show that these fibers are newly formed, Cross striations are seen in fibers. x 190. FIG. 6. Mononucleate single cells liberated from regenerating skeletal muscles (3 days after the jn.jectionj. After selection. Phase contrast. x 270. FIG. 7. Twenty-four hours after plating. The culture consists of mononuleate myogentc cells, fibroblasts, macrophages, and leukocytes. Phase contrast, x 270. FIG. 8. Ten-day-old culture. Muscle fibers developed. Fixed with ethanol, Phase contrast. x 210. FIG. 9. Ten-day-old culture. Note cross striations. Fixed with ethanol. Phase contrast. x 940.
406
DEVELOPMENTAL
BIOLOGY
formed muscle fibers in vitro and no differences were detected concerning their myogenic processes and morphology under the present culture conditions. For this reason, the description will be limited to the differentiating processes, in vitro, of the cells obtained from the leg muscles at 3 days after hypertonic saline injection. When plated, viable cells settled down on culture dishes and began to elongate within 12 hr. At 24 hr of incubation, the cultures consisted of mononucleate, myogenic cells (450/o), fibroblasts (7%), and macrophages and leukocytes (48%) (Fig. 7). Therefore, starting from the muscles of a leg in which regeneration was experimentally induced, approximately 2 x lo” myogenic cells were isolated and successfully cultured under the present conditions. Myogenic cells and fibroblasts started to propagate within 48 hr after plating, whereas macrophages and leukocytes were overgrown by them during the first 3 days of culture; consequently, the cultures consisted of mostly myogenic cells and fibroblasts at 3 days of culture. The ratio of the number of myogenic cells to that of fibroblasts was found to be 1O:l to 10:1.5 from 1 day through 5 days of culture. After 2 days of culture, myogenic cells became gradually smaller in size and rounded and began to assemble, resulting in an oriented pallisade. Among them an intense cell fusion occurred, forming multinucleate myotubes, which increased in number and grew in length during the next 3 days of culture. By 5 days after most mononucleate myogenic plating, cells had been incorporated into multinucleate myotubes. In the center of the myotubes, one or two rows of large, vesicular nuclei possessing one or two nucleoli were observed. With the advancement of myogenesis, the myotubes became longer and thinner. Cross striations were observed in some fibers at 6 days and in all fibers at 10 days of incubation (Figs. 8 and 9). Muscle fibers with cross striations in the present cultures showed only occasional, sponta-
VOLUME
55, 1977
neous contractions. It was found, however, that the use of a culture medium consisting of 89% MEM, 10% horse serum, and 1% chick embryo extract greatly enhanced the muscle fibers’ ability to contract. When transferred to the above culture medium after 4 days of culture, virtually all muscle fibers showed spontaneous contraction at 10 days of culture. An attempt at cell culture was extended to the femoral muscles of adult mice that had not received saline injection. Approximately 5 x lo4 cells per leg were obtained by the same procedure used for the salinetreated muscles. During the process of selective plating, cell number decreased drastically to about one-fortieth of the original cell number. The cells obtained after the selective plating procedure formed muscle fibers under the culture conditions described above. DISCUSSION
Histological findings revealed that the regenerative changes induced by the intramuscular injection of hypertonic saline were similar to those caused by other chemical and physical agents (4). Available data showed that marcain induced synchronous regeneration in the mature skeletal muscles of rats (11). Three features in the present system, however, seem worth mentioning: (a) Hypertonic saline may ultimately become a physiological substance for muscles when it is diluted in tissues; (b) saline injection induced muscular regeneration on a scale large enough to give many myogenic cells without any failure; and (cl histological observation demonstrated that muscle regeneration in the present system proceeded with considerable synchrony. These highly advantageous features for analyzing muscle regeneration have seldom been met by the previously reported experimental means of inducing muscle regeneration in vivo. The injected hypertonic saline may diffuse rapidly into the surrounding tissues, providing a concentration appropriate for the in-
BRIEF
duction of muscle regeneration in certain areas of the tissues and causing an instant injury in the tissues without showing any lag effect. These points may be quite important in attaining the conditions necessary for the synchronous advancement of regenerating processes. In summary, the intramuscular injection of hypertonic saline seems a very simple, yet quite reliable, method to induce regeneration in the skeletal muscles of adult mice, especially for the purpose of obtaining a large enough quantity of cells from the regenerating tissues for the purpose of cell culture studies. The present result showed that approximately 2 x lo” myogenic cells with the potency to differentiate into muscle fibers in vitro were isolated from muscles of a leg of an adult mouse in which regeneration was experimentally induced. Numerous data on the myoblasts from embryos have been accumulated since the pioneering work by Konigsberg (12), but only limited information was available concerning the myogenic cells participating in muscle regeneration in adults. In this sense, the studies on the role of satellite cells in muscle regeneration, in vitro, of a juvenile quail (13) and on the myoblasts isolated from a regenerating tail of a lizard (6) are very important. Mononucleate myogenic cells have been obtained from the explant cultures of skeletal muscles of adult humans (1, 3, 10) and directly from matured skeletal muscles of rats (2). To construct a useful culture system of myogenesis from adult muscles, such features as a large cell yield, the purity of cell population, and the uniformity of cell age are thought to be indispensable prerequisites. The myogenie cell population isolated by the present method may serve as a useful culture system for the analysis of the regenerating
407
NOTES
and differentiating muscles.
phenomena
in skeletal
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