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Characterization of primary human skeletal muscle cells from multiple commercial sources.

Owens J, Moreira K, Bain G - In Vitro Cell. Dev. Biol. Anim. (2013)

Bottom Line: Primary human skeletal muscle cells have recently become available from a number of commercial vendors.However, only limited characterization of these cells has been reported to date.Finally, the myotubes were efficiently infected with recombinant adenovirus, providing a tool for genetic modification.

View Article: PubMed Central - PubMed

Affiliation: Tissue Repair Research Unit, Pfizer, 200 Cambridge Park Drive, Cambridge, MA, 02140, USA, Jane.Owens@Pfizer.com.

ABSTRACT
There is a significant unmet need for safe, anabolic muscle therapies to treat diseases and conditions associated with severe muscle weakness and frailty. The identification of such therapies requires appropriate cell-based screening assays to select compounds for further development using animal models. Primary human skeletal muscle cells have recently become available from a number of commercial vendors. Such cells may be valuable for studying the mechanisms that direct muscle differentiation, and for identifying and characterizing novel therapeutic approaches for the treatment of age- and injury-induced muscle disorders. However, only limited characterization of these cells has been reported to date. Therefore, we have examined four primary human muscle cell preparations from three different vendors for their capacity to differentiate into multinucleated myotubes. Two of the preparations demonstrated robust myotube formation and expressed characteristic markers of muscle differentiation. Furthermore, these myotubes could be induced to undergo morphological atrophy- and hypertrophy-like responses, and atrophy could be blocked with an inhibitor of myostatin signaling, a pathway that is known to negatively regulate muscle mass. Finally, the myotubes were efficiently infected with recombinant adenovirus, providing a tool for genetic modification. Taken together, our results indicate that primary human muscle cells can be a useful system for studying muscle differentiation, and may also provide tools for studying new therapeutic molecules for the treatment of muscle disease.

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ActRIIB-Fc (a myostatin inhibitor) blocks myostatin-induced atrophy in HSMM and SkMDC. MYH2/Hoechst-labeled myotubes differentiated for 3 d and then treated with 1 μg/ml myostatin ± a dilution series of ActRIIB-Fc for 48 h. Representative images of untreated myotubes and those treated with 1 μg/ml Mstn alone and 1 μg/ml Mstn + 50 μg/ml ActRIIB-Fc. Plot shows the mean percent change in myotube area from untreated myotubes ± SEM, n = 15, *p < 0.05 vs 1 μg/ml Mstn using Student’s t test).
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Fig4: ActRIIB-Fc (a myostatin inhibitor) blocks myostatin-induced atrophy in HSMM and SkMDC. MYH2/Hoechst-labeled myotubes differentiated for 3 d and then treated with 1 μg/ml myostatin ± a dilution series of ActRIIB-Fc for 48 h. Representative images of untreated myotubes and those treated with 1 μg/ml Mstn alone and 1 μg/ml Mstn + 50 μg/ml ActRIIB-Fc. Plot shows the mean percent change in myotube area from untreated myotubes ± SEM, n = 15, *p < 0.05 vs 1 μg/ml Mstn using Student’s t test).

Mentions: As shown above, Mstn induced an atrophy-like morphological response in both HSMM and SkMDC where the myotubes showed a substantial decrease in size compared to untreated cultures (Fig. 2A). We and others are working to target myostatin with inhibitors to block its negative regulation of muscle mass as a treatment for frailty. One such inhibitor consists of a fusion between the ligand-binding domain of ActRIIb, the high affinity receptor for Mstn, and the Fc region of immunoglobulin G (ActRIIb-Fc). This molecule is a potent inhibitor of Mstn activity in vivo (Lee et al. 2005). To assess its activity in HSMM and SkMDC, myotubes were treated with Mstn in the presence of various concentrations of ActRIIb-Fc for 48 h. Images of MYH2-positive myotubes were captured and myotube areas were quantified as described above. As expected, the images showed that Mstn alone induced a significant decrease in myotube area, and this atrophy-like response was blocked by ActRIIb-Fc in both cell populations (Fig. 4). The rescue of Mstn-induced atrophy by ActRIIb-Fc was quantified and plotted as the percent change from untreated myotubes (Fig. 4). In both cell populations, 50 μg/ml ActRIIb-Fc induced a significant percent change in area compared to cells treated with Mstn (1 μg/ml) alone (0 μg/ml ActRIIB-Fc). The plots demonstrated that ActRIIB-Fc blocked Mstn-induced atrophy in these cultures, such that the myotube area was significantly different to that measured in cultures treated with Mstn alone. At lower concentrations of ActRIIb-Fc, statistically significant effects were shown in SkMDC cultures only, suggesting that these cells were more sensitive to the rescue effect. Thus, HSMM and SkMDC may be used to identify and characterize inhibitors of myostatin activity.Figure 4.


Characterization of primary human skeletal muscle cells from multiple commercial sources.

Owens J, Moreira K, Bain G - In Vitro Cell. Dev. Biol. Anim. (2013)

ActRIIB-Fc (a myostatin inhibitor) blocks myostatin-induced atrophy in HSMM and SkMDC. MYH2/Hoechst-labeled myotubes differentiated for 3 d and then treated with 1 μg/ml myostatin ± a dilution series of ActRIIB-Fc for 48 h. Representative images of untreated myotubes and those treated with 1 μg/ml Mstn alone and 1 μg/ml Mstn + 50 μg/ml ActRIIB-Fc. Plot shows the mean percent change in myotube area from untreated myotubes ± SEM, n = 15, *p < 0.05 vs 1 μg/ml Mstn using Student’s t test).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824271&req=5

Fig4: ActRIIB-Fc (a myostatin inhibitor) blocks myostatin-induced atrophy in HSMM and SkMDC. MYH2/Hoechst-labeled myotubes differentiated for 3 d and then treated with 1 μg/ml myostatin ± a dilution series of ActRIIB-Fc for 48 h. Representative images of untreated myotubes and those treated with 1 μg/ml Mstn alone and 1 μg/ml Mstn + 50 μg/ml ActRIIB-Fc. Plot shows the mean percent change in myotube area from untreated myotubes ± SEM, n = 15, *p < 0.05 vs 1 μg/ml Mstn using Student’s t test).
Mentions: As shown above, Mstn induced an atrophy-like morphological response in both HSMM and SkMDC where the myotubes showed a substantial decrease in size compared to untreated cultures (Fig. 2A). We and others are working to target myostatin with inhibitors to block its negative regulation of muscle mass as a treatment for frailty. One such inhibitor consists of a fusion between the ligand-binding domain of ActRIIb, the high affinity receptor for Mstn, and the Fc region of immunoglobulin G (ActRIIb-Fc). This molecule is a potent inhibitor of Mstn activity in vivo (Lee et al. 2005). To assess its activity in HSMM and SkMDC, myotubes were treated with Mstn in the presence of various concentrations of ActRIIb-Fc for 48 h. Images of MYH2-positive myotubes were captured and myotube areas were quantified as described above. As expected, the images showed that Mstn alone induced a significant decrease in myotube area, and this atrophy-like response was blocked by ActRIIb-Fc in both cell populations (Fig. 4). The rescue of Mstn-induced atrophy by ActRIIb-Fc was quantified and plotted as the percent change from untreated myotubes (Fig. 4). In both cell populations, 50 μg/ml ActRIIb-Fc induced a significant percent change in area compared to cells treated with Mstn (1 μg/ml) alone (0 μg/ml ActRIIB-Fc). The plots demonstrated that ActRIIB-Fc blocked Mstn-induced atrophy in these cultures, such that the myotube area was significantly different to that measured in cultures treated with Mstn alone. At lower concentrations of ActRIIb-Fc, statistically significant effects were shown in SkMDC cultures only, suggesting that these cells were more sensitive to the rescue effect. Thus, HSMM and SkMDC may be used to identify and characterize inhibitors of myostatin activity.Figure 4.

Bottom Line: Primary human skeletal muscle cells have recently become available from a number of commercial vendors.However, only limited characterization of these cells has been reported to date.Finally, the myotubes were efficiently infected with recombinant adenovirus, providing a tool for genetic modification.

View Article: PubMed Central - PubMed

Affiliation: Tissue Repair Research Unit, Pfizer, 200 Cambridge Park Drive, Cambridge, MA, 02140, USA, Jane.Owens@Pfizer.com.

ABSTRACT
There is a significant unmet need for safe, anabolic muscle therapies to treat diseases and conditions associated with severe muscle weakness and frailty. The identification of such therapies requires appropriate cell-based screening assays to select compounds for further development using animal models. Primary human skeletal muscle cells have recently become available from a number of commercial vendors. Such cells may be valuable for studying the mechanisms that direct muscle differentiation, and for identifying and characterizing novel therapeutic approaches for the treatment of age- and injury-induced muscle disorders. However, only limited characterization of these cells has been reported to date. Therefore, we have examined four primary human muscle cell preparations from three different vendors for their capacity to differentiate into multinucleated myotubes. Two of the preparations demonstrated robust myotube formation and expressed characteristic markers of muscle differentiation. Furthermore, these myotubes could be induced to undergo morphological atrophy- and hypertrophy-like responses, and atrophy could be blocked with an inhibitor of myostatin signaling, a pathway that is known to negatively regulate muscle mass. Finally, the myotubes were efficiently infected with recombinant adenovirus, providing a tool for genetic modification. Taken together, our results indicate that primary human muscle cells can be a useful system for studying muscle differentiation, and may also provide tools for studying new therapeutic molecules for the treatment of muscle disease.

Show MeSH
Related in: MedlinePlus