JOURNAL OF CELLULAR PHYSIOLOGY 146:435441 11991)
Effect of Transforming Growth Factor Beta-1 on Ovine Satellite Cell Proliferation and Fusion MARCIA R. HATHAWAY,* J O A N R. HEMBREE, MARY S. PAMPUSCH AND WILLIAM R. DAYTON Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55 108
We have evaluated the effectof transforming growth factor beta-1 (TGF beta-1)on proliferation and fusion of cultured ovine satellite cells isolated from 5-month-old wether lambs. The isolation and culture protocols were validated by clonal analysis of the original cell preparation and assessment of proliferation and fusion of control cultures. Approximately 85% of the original cells isolated were myogenic as assessed by clonal analysis. The ovine cells doubled approximately every 18 hours during their exponential growth period and achieved a maximum percent fusion of 39.5% after 144 hours in culture. TGF beta-1 inhibited fusion of these cells in a dose-dependentmanner with half-rnaximal inhibition occurring at .08 ng/ml. Maximal inhibition (95% suppression) occurred between .1 and .5 ng/ml. TGF Beta-1 (.05-3.0 ngiml) did not inhibit proliferation of cultured ovine satellite cells in serum-containing medium or in serurn-free defined medium. In contrast, TGF beta-1 did significantly suppress serurn-stimulated proliferation of either porcine or bovine satellite cells that were isolated by using a procedure identical to that used to isolate the ovine satellite cells. Thus, proliferation of ovine satellite cells appears to respond differently to TGF beta-1 than does proliferation of either porcine or bovine satellite cells. Transforming growth factor beta-1 (TGF beta-1) may play an important role in modulating growth in vivo, as it demonstrates multiple, cell-type-specific effects on cultured cells (Roberts and Sporn, 1990; Florini and Magri, 1989). For example, inclusion of TGF beta-1 in the culture media of bronchial or kidney epithelial cells stimulates differentiation and inhibits proliferation (Masui et al., 1986; Fine et al., 1985),while inclusion of TGF beta-1 in the media of murine 3T3 preadi ocytes inhibits differentiation without affecting proli eration (Ignotz and Massague, 1985). TGF beta-1 also inhibits differentiation of cultured myogenic cells as evidenced by reduced expression of differentiation-specific contractile protein gene products and nicotinic acetylcholine receptors, decreased creatine phosphokinase activity, and su pression of myotube formation (Olson et al., 1986). Un ike reports of TGF beta-1’s effect on myogenic cell differentiation, reports of TGF beta-l-induced alteration of cultured myogenic cell proliferation have been contradictory. TGF beta-1 has been reported to have little or no effect on the proliferation of various subclones of the rat L6 cell line (Florini et al., 1986; Massague et al., 1986), the mouse C2 cell line, the RMo cell line (Johnson and Allen, 19901, or the nonfusing mouse BC3H1 cell line (Olson et al., 1986). In contrast, other studies have shown that TGF beta-1 suppresses proliferation of my0 enic cells isolated from neonatal rats (Allen and Box orn, 1987) and porcine embryos (Pampusch et al., 19901, as well as proliferation of the rat L6M1 subclone (Pampusch et al., 1990). Satellite cells have been shown to play an important
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role in the growth of normal muscle (Moss and Leblond, 1971), as well as aiding in the regeneration of injured muscle (reviewed in Carlson and Faulkner, 1983). Consequently, since TGF beta-1 has been shown to suppress both the proliferation and fusion of cultured rat satellite cells (Allen and Boxhorn, 1987; Allen and Boxhorn, 19891, it may influence both postnatal muscle growth and muscle regeneration in this species. To date, the effect of TGF beta-1 on satellite cell proliferation has not been evaluated in any species except the rat (Allen and Boxhorn, 1987; Allen and Boxhorn, 1989). Rat-derived satellite cells may not be an appropriate model for all species, since qualitative differences in response of cultured rat and limb satellite cells to hormonal treatments have been demonstrated (Dodson et al., 1988). Therefore, in order to further investigate the effect of TGF beta-1 on satellite cells, we have evaluated its effect on roliferation and fusion of cultured ovine satellite cel s. As observed in rat satellite cell cultures TGF beta-1 suppresses the fusion of cultured ovine satellite cells. However, contrary to data reported for rat satellite cells, TGF beta-1 does not suppress the proliferation of ovine cells. In contrast, TGF beta-1 does suppress the proliferation of both cultured porcine and bovine satellite cells that were
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Received July 9, 1990; accepted November 16, 1990. *Towhom reprint requestskorrespondenceshould be addressed at 136D Andrew Boss Laboratory, 1354 Eckles Avenue, University of Minnesota, St. Paul, MN 55108.
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isolated by using a procedure identical to that used to isolate ovine satellite cells. MATERIALS AND METHODS Materials TGF beta-1, derived from porcine platelets, was purchased from R&D Systems Inc. (Minneapolis, MN). McCoy’s 5A medium (McCo ’s), horse sera (HS),Earl’s balanced salt solution (EBS ) without CaC1, or MgS04, Deutsch fetuin, bovine fibronectin, transferrin, biotin, sodium selenite, vitamin E, L-glutamine, and antibiotic-antimycotic solution were purchased from Gibco (Grand Island, NY). Pronase (product no. 53702) was purchased from Calbiochem (La Jolla, CA). Basement membrane matrigel, dispase, and bovine fibroblast growth factor were purchased from Collaborative Research (Bedford, MA), Bovine serum albumin (product no. 810281) was purchased from ICN Immunobiologicals (Lisle, IL). Fetal bovine sera (FBS) was purchased from Flow Laboratories Inc. (McLean, VA). Linoleic acid-albumin (product no. L8384), porcine insulin, dexamethasone, vitamin C, myoinositol, sodium pyruvate, and calcium chloride were purchased from Sigma Chemical Co. (St. Louis, MO). Isolation of ovine, porcine, and bovine satellite cells Wether lambs or barrows (approximately 5 months of age) or heifers (approximately 11 months of age) were sacrificed by bolting followed by exsanguination. Using sterile techniques, the portions of the gracilis, semimembranosus, and semitendinosus muscles were dissected out and transported to the cell culture laboratory. Subsequent procedures were conducted in a sterile field under a tissue culture hood. Satellite cells were isolated using a procedure similar to that reported previously for rat and ovine satellite cells (Allen et al., 1984; Dodson et al., 1986). Briefly, excess connective tissue was removed and the muscle was passed through a sterile meat grinder. The ground muscle was incubated with .l% pronase in EBSS for 1 hour at 37°C with frequent mixing. Following incubation the mixture was centrifuged at 1500g for 4 minutes. The supernatant was discarded and the pellet was suspended in phosphate buffered saline (PBS: 140 mM NaC1,l mM KH2P0,, 3 mM KC1,8 mM Na2HP04)and centrifuged at 500g for 10 minutes. The resultant supernatant was then centrifuged at 1500g for 10 minutes to pellet the mononucleated cells. The PBS washings and differential centrifugation were repeated two more times. The resulting mononucleated cell preparation was suspended in McCoy’s containing 10% FBS.
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Culture procedures for ovine satellite cells Ovine satellite cells were plated on 9.62 cm2 culture dishes precoated with basement membrane matrigel (diluted 1:lO in McCoys). Cells were plated in McCoy’s containing 10% FBS and then incubated for 24 hours at 37”C, 5% CO,, 9570air in a water saturated environment. Following this attachment period, cultures were rinsed once with McCoy’s and then fed McCoy’s containing 10% FBS. Culture medium was replaced with McCoy’s containing 10%FBS at 48 and 72 hours. After
96 hours in culture the cells were rinsed with McCoy’s, and then McCoy’s containing 3% horse sera (HS) and 1.5 pg/ml of linoleic acid-albumin was applied. After an additional 48 hours the cells were fixed, stained, and the percent fusion was determined. Determination of fusion percent in non-clonal cultures At the indicated times cells were fixed, Giemsa stained, and examined microscopically to quantify total nuclei and the percentage of nuclei in myotubes. Photographs were taken of random fields using a 35 mm camera and a Zeiss inverted microscope with a 16 X phase-contrast objective. Fusion percent was assessed by determining the ratio of the number of myotube nuclei (a myotube was identified by the presence of 3 or more nuclei within a continuous cell membrane) to total nuclei. A total of 11 fieldslplate and 3 plates per treatment were evaluated. The number of total nuclei evaluated varied depending on the time point and treatment. Relative number of myogenic and nonmyogenic cells in ovine satellite cell preparations Clonal cultures of ovine satellite cells were established by plating at a density of approximately 1 celU.322 cm2 well in McCoys containing 10% FBS. Prior to plating all wells were precoated with basement membrane matrigel (diluted 1:lO with McCoy’s). Cultures were incubated at 37”C, 5% C02, 95% air in a water saturated environment. Following a 24 hour attachment period, the cultures were rinsed once with McCoy’s and then fed McCoy’s containing 10% FBS. Colony growth and fusion were visually monitored on a daily basis for 10 days, after which no additional fusion was observed. The percentage of myogenic cells in the original cell suspension was calculated as the ratio of the number of colonies containing myotubes to the total number of colonies. Effect of TGF beta-1 on ovine satellite cell fusion Ovine satellite cells were plated as described in the ovine satellite culture rocedures. At 24 hours, test media consisting of Mc oy’s containing 10% FBS and various levels of TGF beta-1 were ap lied. In control cultures, TGF beta-1 was replaced wit an equivalent volume of the buffer in which the TGF was dissolved (4 mM HC1 containing 1 mg/ml bovine serum albumin). Since it has been shown that the effect of TGF beta-1 on cultured myoblasts is transitory (Olson et al., 19861, fresh test media were applied at 48 and 72 hours. After 96 hours in culture the cells were rinsed with McCoy’s and then fed McCoy’s containing 3% horse sera, 1.5 yg/ml of linoleic acid-albumin and TGF beta-1 (corresponding to the level in the test media). After an additional 48 hours cells were stained and the percent fusion was determined. The effects of TGF beta-1 on fusion were assessed by evaluating at least 5400 total nuclei for each treatment level. Assay for satellite cell proliferation in serum-containing media Ovine, porcine, or bovine satellite cells were plated and fed as for the ovine satellite cell differentiation
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experiment. At 96 hours the cells were released from the substrata by a 15 minute exposure to 1x dispase at 37°C according to the manufacturer's instructions. The cell suspension was diluted with cold saline and kept on ice until the cell number could be determined using a Coulter counter (Model ZB). Assay for ovine satellite cell proliferation in a defined media Ovine satellite cells were plated as for the ovine satellite cell differentiation experiment. At 24 hours the plating media was removed and replaced with a defined media developed specifically for ovine satellite cells by Dodson and coworkers (1988). The defined media contained various levels of TGF beta-1. In control cultures, TGF beta-1 was replaced with an equivalent volume of the buffer in which the TGF was dissolved (4 mM HC1 containing 1mg/ml bovine serum albumin). Fresh defined media containing TGF beta-1 was applied again at 48 and 72 hours post plating. After 96 hours in culture, the cells were released and counted as described in the assay for satellite cell proliferation in serum-containing medium.
RESULTS Assessment of myogenic and nonmyogenic cell population Clonal analysis is one method that has been used to assess the proportion of myogenic to nonmyogenic cells in primary cultures (Roe et al., 1989; Harper et al., 1986; Yablonka-Reuveni et al., 1987). Consequently, we utilized this procedure to evaluate the relative number of myogenic and nonmyogenic cells present in the ovine satellite cell preparation. When satellite cells from our initial preparations were plated at clonal densities and visually evaluated for fusion, 84.7 ? 2.2% (mean +/- s.e.) of the colonies contained myotubes. This result is the average of 4 experiments, each involving a different ovine satellite cell preparation. A total of 363 colonies were evaluated. Based on the relatively high number of myogenic cells present in the ovine myogenic cell preparation, we concluded that the use of more extensive procedures to further enrich the myogenic cell population was unwarranted. Time course of ovine satellite cell proliferation and fusion Before measuring the effects of TGF beta-1 on the ovine satellite cell cultures, it was necessary to establish the developmental progression of satellite cell proliferation and differentiation under control conditions (Fig. 1). The satellite cells demonstrated a lag period of 48 hours prior to the onset of proliferation, followed by a period of exponential growth between 48 and 96 hours. During the exponential growth period, the doubling time was approximately 18 hours. After 96 hours fresh culture media containing low percent horse sera was applied. At this time, fusion of the cells was initiated and progressed rapidly for an additional 48 hours while proliferation rate was substantially reduced. A maximum of 39.5% fusion was observed at 144 hours. Thus, the ovine satellite cells were capable of attachment, proliferation, and fusion. The bovine and porcine satellite cell cultures also achieved approx-
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Fig. 1. Time course of ovine satellite cell proliferation and fusion. Cells were plated in McCoy's containing 10% FBS. Fresh McCoy's containing 10%FBS was applied at 24,48, and 72 hours. At 96 hours, McCoy's containing 3 9 HS and 1.5 (*g/ml linoleic acid/albumin was applied. Cells were fixed, stained, and microscopically examined to quantify total nuclei and myotube nuclei (Ia) t the indicated times. Stacked bar represents standard error.
imately 40% fusion when cultured under conditions selected to maximize their fusion (data not shown). Effect of TGF beta-1 on ovine satellite cell proliferation in a serum-containing medium To assess the effect of TGF beta-1 on serum-stimulated proliferation of ovine satellite cells, McCoy's containing 10% FBS and various levels of TGF beta-1 were added to the cultures at 24 hours post plating and at 24 hour intervals, for a total of 72 hours of treatment. Cell number was determined at 24, 48, and 72 hours after TGF beta-1 addition to the culture media. The presence of TGF beta-1 in the culture media did not affect serum-stimulated proliferation of ovine satellite cells compared to control cultures after 72 hours of treatment (P < .9) (Fig. 2). Additionally, TGF beta-1 did not alter the proliferation at 24 or 48 hours after its addition (data not shown). The ability of TGF beta-1 to alter ovine satellite cell proliferation after 144 hours of exposure was also assessed by microscopic examination of fixed and stained cultures. In these studies, no difference in total nuclei were detected between TGF beta-1 treated and control cultures (Fig. 3). Effect of TGF beta-1 on the proliferation of ovine satellite cells in a serum-free, defined media Since alpha-2-macroglobulin present in sera has been shown to bind and inactivate TGF beta-1, it is possible that sera may inhibit the ability of TGF beta-1 to su press proliferation (O'Connor-McCourt and Wake ield, et al., 1988). Therefore, the ability of TGF beta-1 to inhibit the proliferation of ovine satellite cells in a serum-free defined medium which did not contain alpha-2-macroglobulin was evaluated. Although the rate of proliferation for all cultures in serum-free, defined media was less than that observed in serumcontaining cultures, TGF beta-1 treatment for as long as 72 hours had no effect ( P < .9) on proliferation rate (Fig. 4).
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Fig. 2. Effect of TGF beta-1 on proliferation of ovine satellite cells in a serum containing medium. Cells were plated in McCoy’s containing 10% FBS. At 24, 48, and 72 hours, fresh test medium consisting of McCoy’s, 10% FBS, and the indicated amount of TGF beta-1 was applied. At 96 hours cells were released from substrata and then counted by using a Coulter counter. Pooled data from 4 assays are shown. Stacked bar represents standard error.
Effect of TGF beta-1 on porcine a n d bovine satellite cell proliferation In order to determine whether the procedure used to isolate ovine satellite cells could be responsible for the inability of TGF beta-1 to suppress their proliferation, the identical isolation protocol was utilized to isolate bovine and porcine satellite cells and the effect of TGF beta-1 on these cells was examined. TGF beta-1 was added to the cultures at 24 hours post plating and at 24 hour intervals, for a total of 72 hours. After 96 hours in culture there was a dose-dependent, TGF beta-1 induced suppression of both porcine (P < .01) and bovine (P < .01) satellite cell proliferation (Fig. 5). In addition to showing that the procedure used to isolate satellite cells does not render them unable to respond to TGF beta-1, these data also establish the activity of the TGF beta-1 used in these studies. Effect of TGF beta-1 on ovine satellite cell fusion The inability of porcine TGF beta-1 to inhibit the proliferation of the cultured ovine satellite cells raised the possibility that these cells were unable to respond to TGF beta-1. To eliminate this possibility we evaluated the ability of TGF beta-1 to inhibit fusion of ovine satellite cells. Figure 3 shows that the porcine TGF beta-1 did suppress fusion of the ovine satellite cells in a dose-dependent manner. Addition of as little as .01 ngiml of TGF beta-1 to the culture medium significantly decreased (P< ,0001) the number of nuclei present in myotubes compared with control cultures, and at .5 ng TGF beta-1 per ml medium, less than 2% of the nuclei were in myotubes.
DISCUSSION Although there are numerous reports of the effect of TGF beta-1 on cultured embryonic myoblasts and cell lines from various species, we are aware of only two reports of the effects of TGF beta-1 on cultured satellite cells. These studies showed that 0.1-0.5 ng/ml TGF
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Fig. 3. Effect of TGF beta-1 on proliferation and fusion of ovine satellite cells. Cells were plated in McCoy’s containing 10%FBS. At 24, 48, and 72 hours, fresh test medium consisting of McCoy’s, 10% FBS, and the indicated amount of TGF beta-1 was applied. At 96 hours, 3% HS and 1.5 kgiml linoleic acidialbumin and a level of TGF Beta-1 corresponding to the level in the original test medium was applied. At 144 hours cells were fixed, stained, and microscopically examined to quantify total nuclei ( E l ) and myotube nuclei (I). Stacked bar represents standard error.
beta-1 resulted in half-maximal suppression of proliferation of satellite cells isolated from rats 3-6 months old (Allen and Boxhorn, 1987; Allen and Boxhorn, 1988). Our data indicate that proliferation of bovine and porcine satellite cells is also significantly inhibited at approximately these same concentrations of TGF beta-1. In contrast, our studies have also shown that TGF beta-1 (0.5-3.0ng/ml) has no effect on proliferation of ovine satellite cells cultured in either serumcontaining medium or a serum-free, defined medium. However, TGF beta-1 did sup less fusion of the ovine satellite cells which suggests t e presence of functional TGF beta-1 receptork). Based on data presented here, as well as on data previously reported (Allen and Boxhorn, 1987), we conclude that ovine satellite cells respond differently to TGF beta-1 than do satellite cells isolated from either the pig, the cow, or the rat. The inability of TGF beta-1 to suppress proliferation of the ovine satellite cells in culture might be due to the protocol utilized to isolate the cells. To test this hypothesis, identical satellite cell isolation preparations were conducted using tissue from both the pig and the cow. In both cases, proliferation of the satellite cells isolated from these species was significantly inhibited when the satellite cell cultures were exposed to very low levels of TGF beta-1. Therefore, it does not appear likely that the isolation protocol itself is accountable for the discrepancy in the results between species. Alpha-2-macroglobulin, a serum protein, has been shown to be responsible for binding both endogenous and exogenous TGF beta-1 (Danielpour and Sporn, 1990; McCaffrey et al., 1989; O’Connor-McCourt and Wakefield, 1987) and may function to scavenge excess TGF beta-1 in the sera (Wakefield et al., 1988).Alpha2-macroglobulin has also been shown to inhibit the binding of exogenous TGF beta-1 to its receptor (Danielpour and Sporn, 1990; O’Connor-McCourt and Wakefield, 1987).Moreover, TGF beta-1 has been reported to have much less activity in serum-su plemented medium (Danielpour and Sporn, 1990; 0’onnor-McCourt
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Fig. 4. Effect of TGF beta-1 on proliferation of ovine satellite cells in a defined media. Defined media consisted of: 10-6M insdin, 25 ngiml FGF, lO-'M dexamethasone, 1 pg/ml serum albumin, 10 pM fetuin, 25 ngiml fibronectin, 5 pgiml transferrin, 2 Wgiml biotin, 3.8 ngiml M vitamin C, 10 ngiml vitamin E, 1 pgiml selenium, 5 x myoinositol, 500 ng/ml L-glutamine, 10 ng/ml linoleic acid, 110 pg/ml Na-pyruvate and 1 mM CaC1,2H20 in McCoys. Cells were plated in McCoy's containing 10% FBS. At 24, 48, and 72 hours, fresh test medium consistin of defined media and the indicated amount of TGF beta-1 was appliei. At 96 hours cells were released from the substrata and then counted by using a Coulter counter. Pooled data from 4 assays are shown. Stacked bar represents standard error.
and Wakefield, 1987). Therefore, the possibility that the alpha-2-macroglobulin present in the serum of the culture medium might be interfering with the inhibitory action of the TGF beta-1 was assessed by exposing the ovine cells to TGF beta-1 in a serum-free, defined media which did not contain serum alpha-2-macroglobulin. The proliferation of ovine satellite cells exposed to TGF beta-1 in this serum-free, defined medium was not altered. Consequently, the presence of serum alpha2-macroglobulin does not appear to be responsible for the inability of TGF beta-1 to inhibit proliferation of ovine satellite cells. This does not rule out the possibility that the ovine cells themselves may secrete alpha-2-macroglobulin into the culture media since other cultured cells have been shown to synthesize and secrete alpha-2-macroglobulin (Shi et al., 1990). The degree to which TGF beta-1 suppresses proliferation of cultured cells has been shown to be slightly affected by the amount of sera in the culture medium (Roberts et al., 1985). Allen and Boxhorn (1987) tested the ability of TGF beta-1 to inhibit adult rat satellite cell proliferation in the presence of 10% horse sera. Therefore, we also tested the ability of TGF beta-1 to inhibit ovine satellite cell proliferation in the presence of 10% horse sera. No inhibition of ovine cell proliferation was detected at any level of TGF beta-1 tested (0.5-3 ng/ml) (data not shown). The frequency of application and duration of treatment could also affect detection of TGF beta-1-induced suppression of myogenic cell proliferation. Allen and Boxhorn (1987) applied test media containing TGF beta-1 to the rat cultures daily for a total of 4 days. Similarly, we applied test media to the ovine cultures daily for either 3 days (Fig. 2) or 4 days (Fig. 3). No difference in cell number between control and treated ovine cultures was observed in either assay. Although
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Fig. 5. Effect of TGF beta-1 on proliferation of (A):bovine and (B): porcine satellite cells in a serum containing medium. Cells were plated in McCoy's containing 10% FBS. At 24,48, and 72 hours, fresh test medium consisting of McCoy's, 10% FBS, and the indicated amount of TGF beta-1 as applied. At 96 hours cells were released from the substrata and then counted by using a Coulter counter. Pooled data from 3 assays are shown for both A and B. T-bars represent standard errors.
the ovine satellite cells undergo a limited number of doublings during the culture eriod due to the lag hase which follows plating, T F beta-1 induced inhiition of proliferation has been reported in adult rat satellite cell cultures which underwent a comparable number of doublin s (Allen and Boxhorn, 1987). Additionally, it should e noted that several levels of TGF beta-1 tested in the ovine satellite cell cultures were higher than those reported necessary for maximum inhibition of proliferation in the rat satellite cell cultures. The activity of the TGF beta-1 utilized in this study was confirmed by its ability to inhibit proliferation of cultured bovine and porcine satellite cells in a dosedependent manner. The assay conditions used to test the ability of TGF beta-1 to inhibit proliferation of the bovine, porcine, and ovine satellite cells were similar, thus eliminating the possibility that differences in assay procedure might affect results. In contrast to the disparity between TGF beta-1's effect on proliferation in the rat and lamb satellite cell cultures, the effect of TGF beta-1 on fusion was similar. TGF beta-1 suppressed fusion in the ovine cultures in a dose-dependent manner with half-maximal inhibition occurring between 0.03 and 0.1 ng TGF beta-liml medium. Similarly, Allen and Boxhorn (1987)reported a half-maximal suppression of fusion in the adult rat myogenic cultures at 0.1 ng/ml TGF beta-1 in the culture media. Since the TGF beta-1 used in these studies was derived from porcine platelets, it is possible that species specificity may be involved in our inability to detect any inhibition of ovine satellite cell proliferation. This
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seems unlikely, however, given the fact that TGF beta-1 is highly conserved with total sequence homology between human, bovine, and porcine mature monomer sequences and a difference of only 1 amino acid between these sequences and that found in the mouse (Sporn et al., 1987). Moreover, porcine TGF beta-1 was capable of suppressing fusion of the ovine satellite cell cultures, as well as proliferation of both bovine and porcine satellite cells. The ovine satellite cell culture system used in this study was characterized by clonal analysis of the original cell preparation and by assessment of proliferation and fusion of cultures. Clonal analysis was used to determine the proportion of myogenic cells present in the ovine satellite cell preparation. Approximately 85% of the cells in the original ovine satellite cell preparation were myogenic. This value is similar to those reported by others utilizing similar procedures to assess relative numbers of myogenic cells in ovine cell preparations (Roe et al., 1988; Harper et al., 1986). Therefore, no additional efforts were made to im rove the ratio of myogenic to nonmyogenic cells by di erential plating or Percoll density gradients (both proven methods in other satellite cell systems). However, it should be noted that the proportion of myogenic cells as determined by clonal analysis of the initial cell preparation cannot be directly compared with the proportion of nuclei contained within myotubes after an extended period of time in culture, for several reasons. YablonkaReuveni and coworkers (1987)have reported that when myogenic cells (as assessed by indirect immunofluorescence techniques using skeletal muscle specific antibodies) were isolated from adult chicken muscle and subsequently cloned, two types of colonies were formed. Most of the colonies formed were large, and of those, over 50% contained areas of actively dividing cells closely associated with myotubes. In contrast, in the few small colonies formed, every cell was terminally differentiated. Thus, it would appear that not all satellite cells are capable of differentiating into myotubes at the same time. The time constraints in culture may not allow all myogenic cells to fuse. Additionally, lating efficiencies may be different in clonal versus Righ density cultures and rates of myogenic and nonmyogenic cell proliferation may be different. The percent fusion determined for control cultures after 144 hours falls within the range reported by others (Dodson et al., 1988; 1990) utilizing cultured ovine satellite cells, even though culture protocols differed. This rovided additional confirmation that our ovine sate lite cell preparation and culture conditions were acceptable. Based on this initial data, it would appear that proliferation of ovine satellite cells may respond differently to TGF beta-1 than proliferation of cultured bovine, porcine, and rat satellite cells. The reason for this difference in response is under investigation. U1timately, the ovine satellite cell cultures may serve as a model system with which to study TGF beta-1 receptors with respect to specific functions.
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ACKNOWLEDGEMENTS The authors would like to express sincere appreciation t o Lori Erikson for technical assistance, to Janet
McNally for coordinating the delivery of lambs, and to Dave Plath for assistance in the abattoir. Thanks is extended to Dr. C. Bingham for assistance in statistical analyses. Published as paper No. 18,771 of the Scientific Journal Series of the Minnesota Agricultural Experiment Station on research conducted under Minnesota Experiment Station projects No. 4816-83 and 4816-80. This work was supported by USDA Competitive Research grant No. 87-CRCR-1-2531.
LITERATURE CITED Allen, R.E., and Boxhorn, L.K. (1989) Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor. J . Cell. Physiol. 138:311-315. Allen, R.E., and Boxhorn, L.K. (1987) Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta. J. Cell. Physiol. 133567-572, Allen, R.E., Dodson, M.V., and Luiten, L.S. (1984) Regulation of skeletal muscle satellite cell proliferation by bovine pituitary fibroblast growth factor. Exp. Cell Res. 152:154-160. Carlson, B.M., and Faulkner, J.A. (1983) The regeneration of skeletal muscle fibers following injury: A review. Med. Sci. Sports Exerc. 15:187-198. Danielpour, D., and Sporn, M.B. (1990) Differential inhibition of transforming growth factor p l and p2 activity by cw2-macroglobulin. J. Biol. Chem. 265:6973-6977. Dodson, M.V., Mathison, B.A., and Mathison, B.D. (1990) Effects of medium and substratum on ovine satellite cell attachment, proliferation and differentiation in vitro. Cell Differ. 29:59-66. Dodson, M.V., Mathison, B.A., Brannon, M.A., Martin, E.L., Wheeler, B.A., and McFarland, D.C. (1988) Comparison of ovine and rat muscle-derived satellite cells: Response to insulin. Tissue Cell 20:909-918. Dodson, M.V., McFarland, D.C., Martin, E.L., and Brannon, M.A. (1986) Isolation of satellite cells from ovine skeletal muscles. J. Tissue Culture Methods 10:233-237. Fine, L.G., Holley, R.W., Nasri, H., and Badie-Dezfooly, B. (1985) BSC-1 growth inhibitor transforms a mitogenic stimulus into a hypertrophic stimulus for renal proximal tubular cells: Relationship to Na'lH' antiport activity. Proc. Natl. Acad. Sci. U.S.A. 82:61636166. Florini, J.R., and Magri, K.A. (1989) Effects of growth factors on myogenic differentiation. Am. J. Physiol. 256:C701-C711. Florini, J.R., Roberts, A.B., Ewton, D.Z., Falen, S.L. Flanders, K.C., and Soorn. M.B. (1986)Transforming prowth factor-B. A verv ootent inhibitor of myoblast differentiation, ivdentical to the differenhation inhibitor secreted by buffalo rat liver cells. J. Biol. Chem. 261:16509-16513. Harper, J.M.M., Soar, F.B., and Buttery, P.J. (1986) Changes in protein metabolism of ovine primary muscle cultures on treatment with growth hormone, insulin, insulin-like growth factor I, or epidermal growth factor. J. Endocrinol. II2:87-96. Ignotz, R.A., and Massague, J . (1985) Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 82:8530-8534. Johnson, S.E., and Allen, R.E. (1990)The effects of bFGF, IGF-1, and TGF-P on RMo skeletal muscle cell proliferation and differentiation. Exp. Cell Res. 187:250-254. Massague, J.,Cheifetz, S., Endo, T., and Nadal-Ginard, B. (1986)Type p transforming owth factor is an inhibitor of myogenic differentiation. Cell Bioy83:8206-8210. Masui, T., Wakefield, L.M., Lechner, J.F., LaVeck, M.A., Sporn, M.B., and Harris, C.C. (1986) Type b transforming growth factor is the primary differentiation inducing factor for normal human bronchial euithelial cells. Proc. Natl. Acad. Sci. U.S.A. 83:2438-2442. McCaffrey, T.A., Falcone, D.J., Brayton, C.F., Agarwal, L.A., Welt, F.G.P., and Weksler, B.B. (1989) J. Cell Biol. 109:441-448. Moss, F.P.. and Leblond, C.P. (1971)Satellite cells as a source of nuclei in muscles of growing rats. Anat. Rec. 170:421436. O'Connor-McCourt, M.D., and Wakefield, L.M. (1987) Latent transforming growth factor-p in serum. J. Biol. Chem. 262:14090-14099. Olson, E.N., Sternberg, E., Hu, J.S., Spizz, G., and Wilcox, C. (1986) Regulation of myogenic differentiation by type p transforming growth factor. J . Cell Biol. 103:1799-1805.
EFFECT OF TGF BETA-1 ON OVINE SATELLITE CELLS Pampusch, M.S., Hembree, J.R., Hathaway, M.R., and Dayton, W.R. (1990) Effect of transforming growth factor (TGF) beta on proliferation of L6 and embryonic porcine myogenic cells. J. Cell. Physiol. 143524528. Roberts, A.B., Anzano, M.A., Wakefield, L.M., Roche, N.S., Stern, D.F., and Sporn, M.B. (1985) Type p transforming growth factor: A bifunctional regulator of cellular growth. Proc. Natl. Acad. Sci. U.S.A. 82:119-123. Roberts, A.B., and Sporn, M.B. (1990) The transforming growth factor-betas. In Peptide Growth Factors and Their Receptors. Handbook of Experimental Pharmacology. Vol. 95. M.B. Sporn and A.B. Roberts, eds. Springer Verlag, Heidelberg, pp. 419-472. Roe, J.A.. Harper. J.M.M., and Buttery, P.J. (1989) Protein metabo-
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lism in ovine orimarv muscle cultures derived from satellite c e l l s Effects of selected peptide hormones and growth factors. J . Endocrinol. 122:565-571. Shi. L.D.. Savona. C.. Gamon. J.. Cochet. C.. Chambaz. E.M.. and Feige, J. (1990)’Transfcbing growth factor-p stimulates the expression of a2-macroglobulin by cultured bovine adrenocortical cells. J. Biol. Chem. 265:2881-2887. Wakefield. L.M.. Smith. D.M.. Flanders. K.C.. and h o r n . M.B. (19881 Latent transforming growth factor-p from human’platelets. J. Biol. Chem. 263:7645-7654. Yablonka-Reuveni, Z.. Quinn. L.S..and Nameroff, M. (1987)Isolation and clonal analysis ofsatellite cells from chicken pectoralis muscle. Dev. Biol. 119:252-259.
Effect of Transforming Growth Factor Beta-1 on Ovine Satellite Cell Proliferation and Fusion MARCIA R. HATHAWAY,* J O A N R. HEMBREE, MARY S. PAMPUSCH AND WILLIAM R. DAYTON Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55 108
We have evaluated the effectof transforming growth factor beta-1 (TGF beta-1)on proliferation and fusion of cultured ovine satellite cells isolated from 5-month-old wether lambs. The isolation and culture protocols were validated by clonal analysis of the original cell preparation and assessment of proliferation and fusion of control cultures. Approximately 85% of the original cells isolated were myogenic as assessed by clonal analysis. The ovine cells doubled approximately every 18 hours during their exponential growth period and achieved a maximum percent fusion of 39.5% after 144 hours in culture. TGF beta-1 inhibited fusion of these cells in a dose-dependentmanner with half-rnaximal inhibition occurring at .08 ng/ml. Maximal inhibition (95% suppression) occurred between .1 and .5 ng/ml. TGF Beta-1 (.05-3.0 ngiml) did not inhibit proliferation of cultured ovine satellite cells in serum-containing medium or in serurn-free defined medium. In contrast, TGF beta-1 did significantly suppress serurn-stimulated proliferation of either porcine or bovine satellite cells that were isolated by using a procedure identical to that used to isolate the ovine satellite cells. Thus, proliferation of ovine satellite cells appears to respond differently to TGF beta-1 than does proliferation of either porcine or bovine satellite cells. Transforming growth factor beta-1 (TGF beta-1) may play an important role in modulating growth in vivo, as it demonstrates multiple, cell-type-specific effects on cultured cells (Roberts and Sporn, 1990; Florini and Magri, 1989). For example, inclusion of TGF beta-1 in the culture media of bronchial or kidney epithelial cells stimulates differentiation and inhibits proliferation (Masui et al., 1986; Fine et al., 1985),while inclusion of TGF beta-1 in the media of murine 3T3 preadi ocytes inhibits differentiation without affecting proli eration (Ignotz and Massague, 1985). TGF beta-1 also inhibits differentiation of cultured myogenic cells as evidenced by reduced expression of differentiation-specific contractile protein gene products and nicotinic acetylcholine receptors, decreased creatine phosphokinase activity, and su pression of myotube formation (Olson et al., 1986). Un ike reports of TGF beta-1’s effect on myogenic cell differentiation, reports of TGF beta-l-induced alteration of cultured myogenic cell proliferation have been contradictory. TGF beta-1 has been reported to have little or no effect on the proliferation of various subclones of the rat L6 cell line (Florini et al., 1986; Massague et al., 1986), the mouse C2 cell line, the RMo cell line (Johnson and Allen, 19901, or the nonfusing mouse BC3H1 cell line (Olson et al., 1986). In contrast, other studies have shown that TGF beta-1 suppresses proliferation of my0 enic cells isolated from neonatal rats (Allen and Box orn, 1987) and porcine embryos (Pampusch et al., 19901, as well as proliferation of the rat L6M1 subclone (Pampusch et al., 1990). Satellite cells have been shown to play an important
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role in the growth of normal muscle (Moss and Leblond, 1971), as well as aiding in the regeneration of injured muscle (reviewed in Carlson and Faulkner, 1983). Consequently, since TGF beta-1 has been shown to suppress both the proliferation and fusion of cultured rat satellite cells (Allen and Boxhorn, 1987; Allen and Boxhorn, 19891, it may influence both postnatal muscle growth and muscle regeneration in this species. To date, the effect of TGF beta-1 on satellite cell proliferation has not been evaluated in any species except the rat (Allen and Boxhorn, 1987; Allen and Boxhorn, 1989). Rat-derived satellite cells may not be an appropriate model for all species, since qualitative differences in response of cultured rat and limb satellite cells to hormonal treatments have been demonstrated (Dodson et al., 1988). Therefore, in order to further investigate the effect of TGF beta-1 on satellite cells, we have evaluated its effect on roliferation and fusion of cultured ovine satellite cel s. As observed in rat satellite cell cultures TGF beta-1 suppresses the fusion of cultured ovine satellite cells. However, contrary to data reported for rat satellite cells, TGF beta-1 does not suppress the proliferation of ovine cells. In contrast, TGF beta-1 does suppress the proliferation of both cultured porcine and bovine satellite cells that were
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Received July 9, 1990; accepted November 16, 1990. *Towhom reprint requestskorrespondenceshould be addressed at 136D Andrew Boss Laboratory, 1354 Eckles Avenue, University of Minnesota, St. Paul, MN 55108.
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isolated by using a procedure identical to that used to isolate ovine satellite cells. MATERIALS AND METHODS Materials TGF beta-1, derived from porcine platelets, was purchased from R&D Systems Inc. (Minneapolis, MN). McCoy’s 5A medium (McCo ’s), horse sera (HS),Earl’s balanced salt solution (EBS ) without CaC1, or MgS04, Deutsch fetuin, bovine fibronectin, transferrin, biotin, sodium selenite, vitamin E, L-glutamine, and antibiotic-antimycotic solution were purchased from Gibco (Grand Island, NY). Pronase (product no. 53702) was purchased from Calbiochem (La Jolla, CA). Basement membrane matrigel, dispase, and bovine fibroblast growth factor were purchased from Collaborative Research (Bedford, MA), Bovine serum albumin (product no. 810281) was purchased from ICN Immunobiologicals (Lisle, IL). Fetal bovine sera (FBS) was purchased from Flow Laboratories Inc. (McLean, VA). Linoleic acid-albumin (product no. L8384), porcine insulin, dexamethasone, vitamin C, myoinositol, sodium pyruvate, and calcium chloride were purchased from Sigma Chemical Co. (St. Louis, MO). Isolation of ovine, porcine, and bovine satellite cells Wether lambs or barrows (approximately 5 months of age) or heifers (approximately 11 months of age) were sacrificed by bolting followed by exsanguination. Using sterile techniques, the portions of the gracilis, semimembranosus, and semitendinosus muscles were dissected out and transported to the cell culture laboratory. Subsequent procedures were conducted in a sterile field under a tissue culture hood. Satellite cells were isolated using a procedure similar to that reported previously for rat and ovine satellite cells (Allen et al., 1984; Dodson et al., 1986). Briefly, excess connective tissue was removed and the muscle was passed through a sterile meat grinder. The ground muscle was incubated with .l% pronase in EBSS for 1 hour at 37°C with frequent mixing. Following incubation the mixture was centrifuged at 1500g for 4 minutes. The supernatant was discarded and the pellet was suspended in phosphate buffered saline (PBS: 140 mM NaC1,l mM KH2P0,, 3 mM KC1,8 mM Na2HP04)and centrifuged at 500g for 10 minutes. The resultant supernatant was then centrifuged at 1500g for 10 minutes to pellet the mononucleated cells. The PBS washings and differential centrifugation were repeated two more times. The resulting mononucleated cell preparation was suspended in McCoy’s containing 10% FBS.
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Culture procedures for ovine satellite cells Ovine satellite cells were plated on 9.62 cm2 culture dishes precoated with basement membrane matrigel (diluted 1:lO in McCoys). Cells were plated in McCoy’s containing 10% FBS and then incubated for 24 hours at 37”C, 5% CO,, 9570air in a water saturated environment. Following this attachment period, cultures were rinsed once with McCoy’s and then fed McCoy’s containing 10% FBS. Culture medium was replaced with McCoy’s containing 10%FBS at 48 and 72 hours. After
96 hours in culture the cells were rinsed with McCoy’s, and then McCoy’s containing 3% horse sera (HS) and 1.5 pg/ml of linoleic acid-albumin was applied. After an additional 48 hours the cells were fixed, stained, and the percent fusion was determined. Determination of fusion percent in non-clonal cultures At the indicated times cells were fixed, Giemsa stained, and examined microscopically to quantify total nuclei and the percentage of nuclei in myotubes. Photographs were taken of random fields using a 35 mm camera and a Zeiss inverted microscope with a 16 X phase-contrast objective. Fusion percent was assessed by determining the ratio of the number of myotube nuclei (a myotube was identified by the presence of 3 or more nuclei within a continuous cell membrane) to total nuclei. A total of 11 fieldslplate and 3 plates per treatment were evaluated. The number of total nuclei evaluated varied depending on the time point and treatment. Relative number of myogenic and nonmyogenic cells in ovine satellite cell preparations Clonal cultures of ovine satellite cells were established by plating at a density of approximately 1 celU.322 cm2 well in McCoys containing 10% FBS. Prior to plating all wells were precoated with basement membrane matrigel (diluted 1:lO with McCoy’s). Cultures were incubated at 37”C, 5% C02, 95% air in a water saturated environment. Following a 24 hour attachment period, the cultures were rinsed once with McCoy’s and then fed McCoy’s containing 10% FBS. Colony growth and fusion were visually monitored on a daily basis for 10 days, after which no additional fusion was observed. The percentage of myogenic cells in the original cell suspension was calculated as the ratio of the number of colonies containing myotubes to the total number of colonies. Effect of TGF beta-1 on ovine satellite cell fusion Ovine satellite cells were plated as described in the ovine satellite culture rocedures. At 24 hours, test media consisting of Mc oy’s containing 10% FBS and various levels of TGF beta-1 were ap lied. In control cultures, TGF beta-1 was replaced wit an equivalent volume of the buffer in which the TGF was dissolved (4 mM HC1 containing 1 mg/ml bovine serum albumin). Since it has been shown that the effect of TGF beta-1 on cultured myoblasts is transitory (Olson et al., 19861, fresh test media were applied at 48 and 72 hours. After 96 hours in culture the cells were rinsed with McCoy’s and then fed McCoy’s containing 3% horse sera, 1.5 yg/ml of linoleic acid-albumin and TGF beta-1 (corresponding to the level in the test media). After an additional 48 hours cells were stained and the percent fusion was determined. The effects of TGF beta-1 on fusion were assessed by evaluating at least 5400 total nuclei for each treatment level. Assay for satellite cell proliferation in serum-containing media Ovine, porcine, or bovine satellite cells were plated and fed as for the ovine satellite cell differentiation
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experiment. At 96 hours the cells were released from the substrata by a 15 minute exposure to 1x dispase at 37°C according to the manufacturer's instructions. The cell suspension was diluted with cold saline and kept on ice until the cell number could be determined using a Coulter counter (Model ZB). Assay for ovine satellite cell proliferation in a defined media Ovine satellite cells were plated as for the ovine satellite cell differentiation experiment. At 24 hours the plating media was removed and replaced with a defined media developed specifically for ovine satellite cells by Dodson and coworkers (1988). The defined media contained various levels of TGF beta-1. In control cultures, TGF beta-1 was replaced with an equivalent volume of the buffer in which the TGF was dissolved (4 mM HC1 containing 1mg/ml bovine serum albumin). Fresh defined media containing TGF beta-1 was applied again at 48 and 72 hours post plating. After 96 hours in culture, the cells were released and counted as described in the assay for satellite cell proliferation in serum-containing medium.
RESULTS Assessment of myogenic and nonmyogenic cell population Clonal analysis is one method that has been used to assess the proportion of myogenic to nonmyogenic cells in primary cultures (Roe et al., 1989; Harper et al., 1986; Yablonka-Reuveni et al., 1987). Consequently, we utilized this procedure to evaluate the relative number of myogenic and nonmyogenic cells present in the ovine satellite cell preparation. When satellite cells from our initial preparations were plated at clonal densities and visually evaluated for fusion, 84.7 ? 2.2% (mean +/- s.e.) of the colonies contained myotubes. This result is the average of 4 experiments, each involving a different ovine satellite cell preparation. A total of 363 colonies were evaluated. Based on the relatively high number of myogenic cells present in the ovine myogenic cell preparation, we concluded that the use of more extensive procedures to further enrich the myogenic cell population was unwarranted. Time course of ovine satellite cell proliferation and fusion Before measuring the effects of TGF beta-1 on the ovine satellite cell cultures, it was necessary to establish the developmental progression of satellite cell proliferation and differentiation under control conditions (Fig. 1). The satellite cells demonstrated a lag period of 48 hours prior to the onset of proliferation, followed by a period of exponential growth between 48 and 96 hours. During the exponential growth period, the doubling time was approximately 18 hours. After 96 hours fresh culture media containing low percent horse sera was applied. At this time, fusion of the cells was initiated and progressed rapidly for an additional 48 hours while proliferation rate was substantially reduced. A maximum of 39.5% fusion was observed at 144 hours. Thus, the ovine satellite cells were capable of attachment, proliferation, and fusion. The bovine and porcine satellite cell cultures also achieved approx-
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Fig. 1. Time course of ovine satellite cell proliferation and fusion. Cells were plated in McCoy's containing 10% FBS. Fresh McCoy's containing 10%FBS was applied at 24,48, and 72 hours. At 96 hours, McCoy's containing 3 9 HS and 1.5 (*g/ml linoleic acid/albumin was applied. Cells were fixed, stained, and microscopically examined to quantify total nuclei and myotube nuclei (Ia) t the indicated times. Stacked bar represents standard error.
imately 40% fusion when cultured under conditions selected to maximize their fusion (data not shown). Effect of TGF beta-1 on ovine satellite cell proliferation in a serum-containing medium To assess the effect of TGF beta-1 on serum-stimulated proliferation of ovine satellite cells, McCoy's containing 10% FBS and various levels of TGF beta-1 were added to the cultures at 24 hours post plating and at 24 hour intervals, for a total of 72 hours of treatment. Cell number was determined at 24, 48, and 72 hours after TGF beta-1 addition to the culture media. The presence of TGF beta-1 in the culture media did not affect serum-stimulated proliferation of ovine satellite cells compared to control cultures after 72 hours of treatment (P < .9) (Fig. 2). Additionally, TGF beta-1 did not alter the proliferation at 24 or 48 hours after its addition (data not shown). The ability of TGF beta-1 to alter ovine satellite cell proliferation after 144 hours of exposure was also assessed by microscopic examination of fixed and stained cultures. In these studies, no difference in total nuclei were detected between TGF beta-1 treated and control cultures (Fig. 3). Effect of TGF beta-1 on the proliferation of ovine satellite cells in a serum-free, defined media Since alpha-2-macroglobulin present in sera has been shown to bind and inactivate TGF beta-1, it is possible that sera may inhibit the ability of TGF beta-1 to su press proliferation (O'Connor-McCourt and Wake ield, et al., 1988). Therefore, the ability of TGF beta-1 to inhibit the proliferation of ovine satellite cells in a serum-free defined medium which did not contain alpha-2-macroglobulin was evaluated. Although the rate of proliferation for all cultures in serum-free, defined media was less than that observed in serumcontaining cultures, TGF beta-1 treatment for as long as 72 hours had no effect ( P < .9) on proliferation rate (Fig. 4).
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Fig. 2. Effect of TGF beta-1 on proliferation of ovine satellite cells in a serum containing medium. Cells were plated in McCoy’s containing 10% FBS. At 24, 48, and 72 hours, fresh test medium consisting of McCoy’s, 10% FBS, and the indicated amount of TGF beta-1 was applied. At 96 hours cells were released from substrata and then counted by using a Coulter counter. Pooled data from 4 assays are shown. Stacked bar represents standard error.
Effect of TGF beta-1 on porcine a n d bovine satellite cell proliferation In order to determine whether the procedure used to isolate ovine satellite cells could be responsible for the inability of TGF beta-1 to suppress their proliferation, the identical isolation protocol was utilized to isolate bovine and porcine satellite cells and the effect of TGF beta-1 on these cells was examined. TGF beta-1 was added to the cultures at 24 hours post plating and at 24 hour intervals, for a total of 72 hours. After 96 hours in culture there was a dose-dependent, TGF beta-1 induced suppression of both porcine (P < .01) and bovine (P < .01) satellite cell proliferation (Fig. 5). In addition to showing that the procedure used to isolate satellite cells does not render them unable to respond to TGF beta-1, these data also establish the activity of the TGF beta-1 used in these studies. Effect of TGF beta-1 on ovine satellite cell fusion The inability of porcine TGF beta-1 to inhibit the proliferation of the cultured ovine satellite cells raised the possibility that these cells were unable to respond to TGF beta-1. To eliminate this possibility we evaluated the ability of TGF beta-1 to inhibit fusion of ovine satellite cells. Figure 3 shows that the porcine TGF beta-1 did suppress fusion of the ovine satellite cells in a dose-dependent manner. Addition of as little as .01 ngiml of TGF beta-1 to the culture medium significantly decreased (P< ,0001) the number of nuclei present in myotubes compared with control cultures, and at .5 ng TGF beta-1 per ml medium, less than 2% of the nuclei were in myotubes.
DISCUSSION Although there are numerous reports of the effect of TGF beta-1 on cultured embryonic myoblasts and cell lines from various species, we are aware of only two reports of the effects of TGF beta-1 on cultured satellite cells. These studies showed that 0.1-0.5 ng/ml TGF
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Fig. 3. Effect of TGF beta-1 on proliferation and fusion of ovine satellite cells. Cells were plated in McCoy’s containing 10%FBS. At 24, 48, and 72 hours, fresh test medium consisting of McCoy’s, 10% FBS, and the indicated amount of TGF beta-1 was applied. At 96 hours, 3% HS and 1.5 kgiml linoleic acidialbumin and a level of TGF Beta-1 corresponding to the level in the original test medium was applied. At 144 hours cells were fixed, stained, and microscopically examined to quantify total nuclei ( E l ) and myotube nuclei (I). Stacked bar represents standard error.
beta-1 resulted in half-maximal suppression of proliferation of satellite cells isolated from rats 3-6 months old (Allen and Boxhorn, 1987; Allen and Boxhorn, 1988). Our data indicate that proliferation of bovine and porcine satellite cells is also significantly inhibited at approximately these same concentrations of TGF beta-1. In contrast, our studies have also shown that TGF beta-1 (0.5-3.0ng/ml) has no effect on proliferation of ovine satellite cells cultured in either serumcontaining medium or a serum-free, defined medium. However, TGF beta-1 did sup less fusion of the ovine satellite cells which suggests t e presence of functional TGF beta-1 receptork). Based on data presented here, as well as on data previously reported (Allen and Boxhorn, 1987), we conclude that ovine satellite cells respond differently to TGF beta-1 than do satellite cells isolated from either the pig, the cow, or the rat. The inability of TGF beta-1 to suppress proliferation of the ovine satellite cells in culture might be due to the protocol utilized to isolate the cells. To test this hypothesis, identical satellite cell isolation preparations were conducted using tissue from both the pig and the cow. In both cases, proliferation of the satellite cells isolated from these species was significantly inhibited when the satellite cell cultures were exposed to very low levels of TGF beta-1. Therefore, it does not appear likely that the isolation protocol itself is accountable for the discrepancy in the results between species. Alpha-2-macroglobulin, a serum protein, has been shown to be responsible for binding both endogenous and exogenous TGF beta-1 (Danielpour and Sporn, 1990; McCaffrey et al., 1989; O’Connor-McCourt and Wakefield, 1987) and may function to scavenge excess TGF beta-1 in the sera (Wakefield et al., 1988).Alpha2-macroglobulin has also been shown to inhibit the binding of exogenous TGF beta-1 to its receptor (Danielpour and Sporn, 1990; O’Connor-McCourt and Wakefield, 1987).Moreover, TGF beta-1 has been reported to have much less activity in serum-su plemented medium (Danielpour and Sporn, 1990; 0’onnor-McCourt
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Fig. 4. Effect of TGF beta-1 on proliferation of ovine satellite cells in a defined media. Defined media consisted of: 10-6M insdin, 25 ngiml FGF, lO-'M dexamethasone, 1 pg/ml serum albumin, 10 pM fetuin, 25 ngiml fibronectin, 5 pgiml transferrin, 2 Wgiml biotin, 3.8 ngiml M vitamin C, 10 ngiml vitamin E, 1 pgiml selenium, 5 x myoinositol, 500 ng/ml L-glutamine, 10 ng/ml linoleic acid, 110 pg/ml Na-pyruvate and 1 mM CaC1,2H20 in McCoys. Cells were plated in McCoy's containing 10% FBS. At 24, 48, and 72 hours, fresh test medium consistin of defined media and the indicated amount of TGF beta-1 was appliei. At 96 hours cells were released from the substrata and then counted by using a Coulter counter. Pooled data from 4 assays are shown. Stacked bar represents standard error.
and Wakefield, 1987). Therefore, the possibility that the alpha-2-macroglobulin present in the serum of the culture medium might be interfering with the inhibitory action of the TGF beta-1 was assessed by exposing the ovine cells to TGF beta-1 in a serum-free, defined media which did not contain serum alpha-2-macroglobulin. The proliferation of ovine satellite cells exposed to TGF beta-1 in this serum-free, defined medium was not altered. Consequently, the presence of serum alpha2-macroglobulin does not appear to be responsible for the inability of TGF beta-1 to inhibit proliferation of ovine satellite cells. This does not rule out the possibility that the ovine cells themselves may secrete alpha-2-macroglobulin into the culture media since other cultured cells have been shown to synthesize and secrete alpha-2-macroglobulin (Shi et al., 1990). The degree to which TGF beta-1 suppresses proliferation of cultured cells has been shown to be slightly affected by the amount of sera in the culture medium (Roberts et al., 1985). Allen and Boxhorn (1987) tested the ability of TGF beta-1 to inhibit adult rat satellite cell proliferation in the presence of 10% horse sera. Therefore, we also tested the ability of TGF beta-1 to inhibit ovine satellite cell proliferation in the presence of 10% horse sera. No inhibition of ovine cell proliferation was detected at any level of TGF beta-1 tested (0.5-3 ng/ml) (data not shown). The frequency of application and duration of treatment could also affect detection of TGF beta-1-induced suppression of myogenic cell proliferation. Allen and Boxhorn (1987) applied test media containing TGF beta-1 to the rat cultures daily for a total of 4 days. Similarly, we applied test media to the ovine cultures daily for either 3 days (Fig. 2) or 4 days (Fig. 3). No difference in cell number between control and treated ovine cultures was observed in either assay. Although
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Fig. 5. Effect of TGF beta-1 on proliferation of (A):bovine and (B): porcine satellite cells in a serum containing medium. Cells were plated in McCoy's containing 10% FBS. At 24,48, and 72 hours, fresh test medium consisting of McCoy's, 10% FBS, and the indicated amount of TGF beta-1 as applied. At 96 hours cells were released from the substrata and then counted by using a Coulter counter. Pooled data from 3 assays are shown for both A and B. T-bars represent standard errors.
the ovine satellite cells undergo a limited number of doublings during the culture eriod due to the lag hase which follows plating, T F beta-1 induced inhiition of proliferation has been reported in adult rat satellite cell cultures which underwent a comparable number of doublin s (Allen and Boxhorn, 1987). Additionally, it should e noted that several levels of TGF beta-1 tested in the ovine satellite cell cultures were higher than those reported necessary for maximum inhibition of proliferation in the rat satellite cell cultures. The activity of the TGF beta-1 utilized in this study was confirmed by its ability to inhibit proliferation of cultured bovine and porcine satellite cells in a dosedependent manner. The assay conditions used to test the ability of TGF beta-1 to inhibit proliferation of the bovine, porcine, and ovine satellite cells were similar, thus eliminating the possibility that differences in assay procedure might affect results. In contrast to the disparity between TGF beta-1's effect on proliferation in the rat and lamb satellite cell cultures, the effect of TGF beta-1 on fusion was similar. TGF beta-1 suppressed fusion in the ovine cultures in a dose-dependent manner with half-maximal inhibition occurring between 0.03 and 0.1 ng TGF beta-liml medium. Similarly, Allen and Boxhorn (1987)reported a half-maximal suppression of fusion in the adult rat myogenic cultures at 0.1 ng/ml TGF beta-1 in the culture media. Since the TGF beta-1 used in these studies was derived from porcine platelets, it is possible that species specificity may be involved in our inability to detect any inhibition of ovine satellite cell proliferation. This
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seems unlikely, however, given the fact that TGF beta-1 is highly conserved with total sequence homology between human, bovine, and porcine mature monomer sequences and a difference of only 1 amino acid between these sequences and that found in the mouse (Sporn et al., 1987). Moreover, porcine TGF beta-1 was capable of suppressing fusion of the ovine satellite cell cultures, as well as proliferation of both bovine and porcine satellite cells. The ovine satellite cell culture system used in this study was characterized by clonal analysis of the original cell preparation and by assessment of proliferation and fusion of cultures. Clonal analysis was used to determine the proportion of myogenic cells present in the ovine satellite cell preparation. Approximately 85% of the cells in the original ovine satellite cell preparation were myogenic. This value is similar to those reported by others utilizing similar procedures to assess relative numbers of myogenic cells in ovine cell preparations (Roe et al., 1988; Harper et al., 1986). Therefore, no additional efforts were made to im rove the ratio of myogenic to nonmyogenic cells by di erential plating or Percoll density gradients (both proven methods in other satellite cell systems). However, it should be noted that the proportion of myogenic cells as determined by clonal analysis of the initial cell preparation cannot be directly compared with the proportion of nuclei contained within myotubes after an extended period of time in culture, for several reasons. YablonkaReuveni and coworkers (1987)have reported that when myogenic cells (as assessed by indirect immunofluorescence techniques using skeletal muscle specific antibodies) were isolated from adult chicken muscle and subsequently cloned, two types of colonies were formed. Most of the colonies formed were large, and of those, over 50% contained areas of actively dividing cells closely associated with myotubes. In contrast, in the few small colonies formed, every cell was terminally differentiated. Thus, it would appear that not all satellite cells are capable of differentiating into myotubes at the same time. The time constraints in culture may not allow all myogenic cells to fuse. Additionally, lating efficiencies may be different in clonal versus Righ density cultures and rates of myogenic and nonmyogenic cell proliferation may be different. The percent fusion determined for control cultures after 144 hours falls within the range reported by others (Dodson et al., 1988; 1990) utilizing cultured ovine satellite cells, even though culture protocols differed. This rovided additional confirmation that our ovine sate lite cell preparation and culture conditions were acceptable. Based on this initial data, it would appear that proliferation of ovine satellite cells may respond differently to TGF beta-1 than proliferation of cultured bovine, porcine, and rat satellite cells. The reason for this difference in response is under investigation. U1timately, the ovine satellite cell cultures may serve as a model system with which to study TGF beta-1 receptors with respect to specific functions.
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ACKNOWLEDGEMENTS The authors would like to express sincere appreciation t o Lori Erikson for technical assistance, to Janet
McNally for coordinating the delivery of lambs, and to Dave Plath for assistance in the abattoir. Thanks is extended to Dr. C. Bingham for assistance in statistical analyses. Published as paper No. 18,771 of the Scientific Journal Series of the Minnesota Agricultural Experiment Station on research conducted under Minnesota Experiment Station projects No. 4816-83 and 4816-80. This work was supported by USDA Competitive Research grant No. 87-CRCR-1-2531.
LITERATURE CITED Allen, R.E., and Boxhorn, L.K. (1989) Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor. J . Cell. Physiol. 138:311-315. Allen, R.E., and Boxhorn, L.K. (1987) Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta. J. Cell. Physiol. 133567-572, Allen, R.E., Dodson, M.V., and Luiten, L.S. (1984) Regulation of skeletal muscle satellite cell proliferation by bovine pituitary fibroblast growth factor. Exp. Cell Res. 152:154-160. Carlson, B.M., and Faulkner, J.A. (1983) The regeneration of skeletal muscle fibers following injury: A review. Med. Sci. Sports Exerc. 15:187-198. Danielpour, D., and Sporn, M.B. (1990) Differential inhibition of transforming growth factor p l and p2 activity by cw2-macroglobulin. J. Biol. Chem. 265:6973-6977. Dodson, M.V., Mathison, B.A., and Mathison, B.D. (1990) Effects of medium and substratum on ovine satellite cell attachment, proliferation and differentiation in vitro. Cell Differ. 29:59-66. Dodson, M.V., Mathison, B.A., Brannon, M.A., Martin, E.L., Wheeler, B.A., and McFarland, D.C. (1988) Comparison of ovine and rat muscle-derived satellite cells: Response to insulin. Tissue Cell 20:909-918. Dodson, M.V., McFarland, D.C., Martin, E.L., and Brannon, M.A. (1986) Isolation of satellite cells from ovine skeletal muscles. J. Tissue Culture Methods 10:233-237. Fine, L.G., Holley, R.W., Nasri, H., and Badie-Dezfooly, B. (1985) BSC-1 growth inhibitor transforms a mitogenic stimulus into a hypertrophic stimulus for renal proximal tubular cells: Relationship to Na'lH' antiport activity. Proc. Natl. Acad. Sci. U.S.A. 82:61636166. Florini, J.R., and Magri, K.A. (1989) Effects of growth factors on myogenic differentiation. Am. J. Physiol. 256:C701-C711. Florini, J.R., Roberts, A.B., Ewton, D.Z., Falen, S.L. Flanders, K.C., and Soorn. M.B. (1986)Transforming prowth factor-B. A verv ootent inhibitor of myoblast differentiation, ivdentical to the differenhation inhibitor secreted by buffalo rat liver cells. J. Biol. Chem. 261:16509-16513. Harper, J.M.M., Soar, F.B., and Buttery, P.J. (1986) Changes in protein metabolism of ovine primary muscle cultures on treatment with growth hormone, insulin, insulin-like growth factor I, or epidermal growth factor. J. Endocrinol. II2:87-96. Ignotz, R.A., and Massague, J . (1985) Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 82:8530-8534. Johnson, S.E., and Allen, R.E. (1990)The effects of bFGF, IGF-1, and TGF-P on RMo skeletal muscle cell proliferation and differentiation. Exp. Cell Res. 187:250-254. Massague, J.,Cheifetz, S., Endo, T., and Nadal-Ginard, B. (1986)Type p transforming owth factor is an inhibitor of myogenic differentiation. Cell Bioy83:8206-8210. Masui, T., Wakefield, L.M., Lechner, J.F., LaVeck, M.A., Sporn, M.B., and Harris, C.C. (1986) Type b transforming growth factor is the primary differentiation inducing factor for normal human bronchial euithelial cells. Proc. Natl. Acad. Sci. U.S.A. 83:2438-2442. McCaffrey, T.A., Falcone, D.J., Brayton, C.F., Agarwal, L.A., Welt, F.G.P., and Weksler, B.B. (1989) J. Cell Biol. 109:441-448. Moss, F.P.. and Leblond, C.P. (1971)Satellite cells as a source of nuclei in muscles of growing rats. Anat. Rec. 170:421436. O'Connor-McCourt, M.D., and Wakefield, L.M. (1987) Latent transforming growth factor-p in serum. J. Biol. Chem. 262:14090-14099. Olson, E.N., Sternberg, E., Hu, J.S., Spizz, G., and Wilcox, C. (1986) Regulation of myogenic differentiation by type p transforming growth factor. J . Cell Biol. 103:1799-1805.
EFFECT OF TGF BETA-1 ON OVINE SATELLITE CELLS Pampusch, M.S., Hembree, J.R., Hathaway, M.R., and Dayton, W.R. (1990) Effect of transforming growth factor (TGF) beta on proliferation of L6 and embryonic porcine myogenic cells. J. Cell. Physiol. 143524528. Roberts, A.B., Anzano, M.A., Wakefield, L.M., Roche, N.S., Stern, D.F., and Sporn, M.B. (1985) Type p transforming growth factor: A bifunctional regulator of cellular growth. Proc. Natl. Acad. Sci. U.S.A. 82:119-123. Roberts, A.B., and Sporn, M.B. (1990) The transforming growth factor-betas. In Peptide Growth Factors and Their Receptors. Handbook of Experimental Pharmacology. Vol. 95. M.B. Sporn and A.B. Roberts, eds. Springer Verlag, Heidelberg, pp. 419-472. Roe, J.A.. Harper. J.M.M., and Buttery, P.J. (1989) Protein metabo-
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lism in ovine orimarv muscle cultures derived from satellite c e l l s Effects of selected peptide hormones and growth factors. J . Endocrinol. 122:565-571. Shi. L.D.. Savona. C.. Gamon. J.. Cochet. C.. Chambaz. E.M.. and Feige, J. (1990)’Transfcbing growth factor-p stimulates the expression of a2-macroglobulin by cultured bovine adrenocortical cells. J. Biol. Chem. 265:2881-2887. Wakefield. L.M.. Smith. D.M.. Flanders. K.C.. and h o r n . M.B. (19881 Latent transforming growth factor-p from human’platelets. J. Biol. Chem. 263:7645-7654. Yablonka-Reuveni, Z.. Quinn. L.S..and Nameroff, M. (1987)Isolation and clonal analysis ofsatellite cells from chicken pectoralis muscle. Dev. Biol. 119:252-259.
