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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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Related in: MedlinePlus

Infection of HSMM and SkMDC with adenovirus. Representative images of MYH2/Hoechst-labeled myotubes differentiated for 2 d and then infected with adeno-GFP at 500 MOI. MYH2 stained red (Not infected images) in this experiment and adeno-GFP is green. The overlap results in a yellow/orange color which demonstrates expression of GFP in MYH2-positive myotubes (Ad-GFP images). (Magnification ×10; scale bar = 100 microns).
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Fig5: Infection of HSMM and SkMDC with adenovirus. Representative images of MYH2/Hoechst-labeled myotubes differentiated for 2 d and then infected with adeno-GFP at 500 MOI. MYH2 stained red (Not infected images) in this experiment and adeno-GFP is green. The overlap results in a yellow/orange color which demonstrates expression of GFP in MYH2-positive myotubes (Ad-GFP images). (Magnification ×10; scale bar = 100 microns).

Mentions: To investigate an alternative method of gene delivery to HSMM and SkMDC, an adenovirus expressing GFP was used to infect differentiated myotubes. Myotubes were identified by staining with anti-MYH2 and detected with Alexa Fluor 555 goat antimouse IgG secondary antibody (red), while the infected cells were detected by GFP fluorescence (green). Infected myotubes were orange (red plus green), uninfected myotubes were red, and infected, undifferentiated cells were green. Although not quantified, it was noted that almost all the myotubes were orange. In addition, most of the undifferentiated (non-myotube) cells in the cultures were also successfully infected, displaying mostly GFP-positive cells (Fig. 5). Finally, undifferentiated cells were also effectively infected by the adenovirus, although the infection efficiency varied from experiment to experiment from ∼30% to 80%, quantified by visual inspection only (data not shown). However, infection of the undifferentiated cells slowed down myotube differentiation by about 3–4 d (data not shown). In general, adenoviral infection was found to be the most efficient method for gene delivery into both cell populations.Figure 5.


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

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

Infection of HSMM and SkMDC with adenovirus. Representative images of MYH2/Hoechst-labeled myotubes differentiated for 2 d and then infected with adeno-GFP at 500 MOI. MYH2 stained red (Not infected images) in this experiment and adeno-GFP is green. The overlap results in a yellow/orange color which demonstrates expression of GFP in MYH2-positive myotubes (Ad-GFP images). (Magnification ×10; scale bar = 100 microns).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Infection of HSMM and SkMDC with adenovirus. Representative images of MYH2/Hoechst-labeled myotubes differentiated for 2 d and then infected with adeno-GFP at 500 MOI. MYH2 stained red (Not infected images) in this experiment and adeno-GFP is green. The overlap results in a yellow/orange color which demonstrates expression of GFP in MYH2-positive myotubes (Ad-GFP images). (Magnification ×10; scale bar = 100 microns).
Mentions: To investigate an alternative method of gene delivery to HSMM and SkMDC, an adenovirus expressing GFP was used to infect differentiated myotubes. Myotubes were identified by staining with anti-MYH2 and detected with Alexa Fluor 555 goat antimouse IgG secondary antibody (red), while the infected cells were detected by GFP fluorescence (green). Infected myotubes were orange (red plus green), uninfected myotubes were red, and infected, undifferentiated cells were green. Although not quantified, it was noted that almost all the myotubes were orange. In addition, most of the undifferentiated (non-myotube) cells in the cultures were also successfully infected, displaying mostly GFP-positive cells (Fig. 5). Finally, undifferentiated cells were also effectively infected by the adenovirus, although the infection efficiency varied from experiment to experiment from ∼30% to 80%, quantified by visual inspection only (data not shown). However, infection of the undifferentiated cells slowed down myotube differentiation by about 3–4 d (data not shown). In general, adenoviral infection was found to be the most efficient method for gene delivery into both cell populations.Figure 5.

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