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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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HSMM and SkMDC differentiated into multinucleated Myosin Heavy Chain-2 (MYH2)-positive myotubes. (A) Representative phase contrast and dark-field images of MYH2-immunostained cells (green), with Hoechst-labeled nuclei (blue) after 1 d in growth medium (phase contrast images) or 3 d in differentiation medium (dark-field images; magnification ×10; scale bar = 100 microns). (B) Skeletal muscle cell marker gene expression by RT-PCR. Plots show gene expression of early (MYOD, Mef2C) and late (MYOG, TNNT, and MYH2) muscle cell markers, normalized to B2M gene expression, relative to expression levels at day 0 (time at which differentiation media was added to the cells); *p < 0.05 vs. day 0 using Student’s t test.
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Fig1: HSMM and SkMDC differentiated into multinucleated Myosin Heavy Chain-2 (MYH2)-positive myotubes. (A) Representative phase contrast and dark-field images of MYH2-immunostained cells (green), with Hoechst-labeled nuclei (blue) after 1 d in growth medium (phase contrast images) or 3 d in differentiation medium (dark-field images; magnification ×10; scale bar = 100 microns). (B) Skeletal muscle cell marker gene expression by RT-PCR. Plots show gene expression of early (MYOD, Mef2C) and late (MYOG, TNNT, and MYH2) muscle cell markers, normalized to B2M gene expression, relative to expression levels at day 0 (time at which differentiation media was added to the cells); *p < 0.05 vs. day 0 using Student’s t test.

Mentions: Both HSMM and SkMDC differentiated into myosin heavy chain (MYH2)-positive multinucleated myotubes within 3 d when cultured on polystyrene cell culture plates in differentiation medium (Fig. 1A). In general, the undifferentiated cells cultured in growth medium did not express MYH2, although occasional MYH2-positive cells were seen in growing cultures, possibly resulting from cell confluence associated contact-dependent cell differentiation (see SkMDC in Fig. 1A). To minimize this issue, care was taken to subculture growing cultures of cells before they became confluent. It was noted that the organization of nuclei within the myotubes was different between HSMM and SkMDC. In HSMM myotubes, the nuclei tend to be arranged as singlets, or small groups, in linear arrays. In contrast, the nuclei in SkMDC myotubes were often clustered together in large groups (Fig. 1A). This difference may be caused by differences in the mechanisms of cell fusion during myotube generation, or by differences in the regulation of nuclear dynamics in the myotubes, or other unknown factors. To further characterize the differentiation process of HSMM and SkMDC, RNA was prepared from cells harvested on day 0 through day 6 of culture in differentiation medium. Gene expression analyses were performed using quantitative RT-PCR for various markers of muscle cell differentiation, (Fig. 1B). In all cases, gene expression levels were normalized to beta-2 microglobulin (B2M) expression levels in order to assess changes in relative expression. MyoD and Mef2C, transcription factors that play important roles in specifying the myogenic lineage, represent two early markers of myoblast differentiation into myotubes (Megeney and Rudnicki 1995; Black and Olson 1998; Zammit et al. 2006). In both HSMM and SkMDC, there was a transient, modest upregulation of MyoD at day 1 (although it was statistically significant for HSMM only) which returned to baseline levels by day 2. Mef2C was also upregulated by day 1 in both cell populations, reaching statistical significance by day 2, but unlike MyoD, the expression of this gene remained elevated throughout the 6-d culture period. Myogenin (MYOG), Troponin T (TNNT), and MYH2 represent late markers of muscle cell differentiation (Burattini et al. 2004; Ozernyuk and Balan 2007). Similar expression trends with these markers were seen in HSMM and SkMDC cultures. MYOG was upregulated approximately tenfold by day 1 of differentiation and that expression level remained significantly elevated from day 2 to day 6 of culture. TNNT was significantly upregulated by day 1 of differentiation in HSMM and by day 2 of differentiation for SkMDC. TNNT expression remained at least tenfold upregulated through day 6 of differentiation in both cell populations. MYH2 was also expressed at least fivefold above control expression levels by day 1 of differentiation for HSMM and by day 2 of differentiation for SkMDC. Then, MYH2 expression reached greater than tenfold above control levels through day 6 of differentiation. Thus, HSMM and SkMDC undergo morphological differentiation in culture into multinucleated myotubes and express characteristic molecular markers of muscle cell differentiation.Figure 1.


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

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

HSMM and SkMDC differentiated into multinucleated Myosin Heavy Chain-2 (MYH2)-positive myotubes. (A) Representative phase contrast and dark-field images of MYH2-immunostained cells (green), with Hoechst-labeled nuclei (blue) after 1 d in growth medium (phase contrast images) or 3 d in differentiation medium (dark-field images; magnification ×10; scale bar = 100 microns). (B) Skeletal muscle cell marker gene expression by RT-PCR. Plots show gene expression of early (MYOD, Mef2C) and late (MYOG, TNNT, and MYH2) muscle cell markers, normalized to B2M gene expression, relative to expression levels at day 0 (time at which differentiation media was added to the cells); *p < 0.05 vs. day 0 using Student’s t test.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824271&req=5

Fig1: HSMM and SkMDC differentiated into multinucleated Myosin Heavy Chain-2 (MYH2)-positive myotubes. (A) Representative phase contrast and dark-field images of MYH2-immunostained cells (green), with Hoechst-labeled nuclei (blue) after 1 d in growth medium (phase contrast images) or 3 d in differentiation medium (dark-field images; magnification ×10; scale bar = 100 microns). (B) Skeletal muscle cell marker gene expression by RT-PCR. Plots show gene expression of early (MYOD, Mef2C) and late (MYOG, TNNT, and MYH2) muscle cell markers, normalized to B2M gene expression, relative to expression levels at day 0 (time at which differentiation media was added to the cells); *p < 0.05 vs. day 0 using Student’s t test.
Mentions: Both HSMM and SkMDC differentiated into myosin heavy chain (MYH2)-positive multinucleated myotubes within 3 d when cultured on polystyrene cell culture plates in differentiation medium (Fig. 1A). In general, the undifferentiated cells cultured in growth medium did not express MYH2, although occasional MYH2-positive cells were seen in growing cultures, possibly resulting from cell confluence associated contact-dependent cell differentiation (see SkMDC in Fig. 1A). To minimize this issue, care was taken to subculture growing cultures of cells before they became confluent. It was noted that the organization of nuclei within the myotubes was different between HSMM and SkMDC. In HSMM myotubes, the nuclei tend to be arranged as singlets, or small groups, in linear arrays. In contrast, the nuclei in SkMDC myotubes were often clustered together in large groups (Fig. 1A). This difference may be caused by differences in the mechanisms of cell fusion during myotube generation, or by differences in the regulation of nuclear dynamics in the myotubes, or other unknown factors. To further characterize the differentiation process of HSMM and SkMDC, RNA was prepared from cells harvested on day 0 through day 6 of culture in differentiation medium. Gene expression analyses were performed using quantitative RT-PCR for various markers of muscle cell differentiation, (Fig. 1B). In all cases, gene expression levels were normalized to beta-2 microglobulin (B2M) expression levels in order to assess changes in relative expression. MyoD and Mef2C, transcription factors that play important roles in specifying the myogenic lineage, represent two early markers of myoblast differentiation into myotubes (Megeney and Rudnicki 1995; Black and Olson 1998; Zammit et al. 2006). In both HSMM and SkMDC, there was a transient, modest upregulation of MyoD at day 1 (although it was statistically significant for HSMM only) which returned to baseline levels by day 2. Mef2C was also upregulated by day 1 in both cell populations, reaching statistical significance by day 2, but unlike MyoD, the expression of this gene remained elevated throughout the 6-d culture period. Myogenin (MYOG), Troponin T (TNNT), and MYH2 represent late markers of muscle cell differentiation (Burattini et al. 2004; Ozernyuk and Balan 2007). Similar expression trends with these markers were seen in HSMM and SkMDC cultures. MYOG was upregulated approximately tenfold by day 1 of differentiation and that expression level remained significantly elevated from day 2 to day 6 of culture. TNNT was significantly upregulated by day 1 of differentiation in HSMM and by day 2 of differentiation for SkMDC. TNNT expression remained at least tenfold upregulated through day 6 of differentiation in both cell populations. MYH2 was also expressed at least fivefold above control expression levels by day 1 of differentiation for HSMM and by day 2 of differentiation for SkMDC. Then, MYH2 expression reached greater than tenfold above control levels through day 6 of differentiation. Thus, HSMM and SkMDC undergo morphological differentiation in culture into multinucleated myotubes and express characteristic molecular markers of muscle cell differentiation.Figure 1.

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