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Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs.

Madden L, Juhas M, Kraus WE, Truskey GA, Bursac N - Elife (2015)

Bottom Line: During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength.In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes.Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, United States.

ABSTRACT
Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7(+) cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

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Characterization of myofiber length and myonuclei number.(A) Representative composite image of a 3-week differentiated myobundle formed using 5% GFP expressing myogenic cells to visualize individual myofibers. Scale bar = 500 µm. (B) The average length of GFP+ myofibers as a function of differentiation time (20–50 myofibers per bundle, n = 4–6 myobundles per time point). (C) Histogram of myonuclei number per GFP+ myofiber in 3-week myobundles with average and median myonuclei numbers of 7 ± 3.6 and 6, respectively (n = 127 myofibers from 6 myobundles).DOI:http://dx.doi.org/10.7554/eLife.04885.007
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fig1s4: Characterization of myofiber length and myonuclei number.(A) Representative composite image of a 3-week differentiated myobundle formed using 5% GFP expressing myogenic cells to visualize individual myofibers. Scale bar = 500 µm. (B) The average length of GFP+ myofibers as a function of differentiation time (20–50 myofibers per bundle, n = 4–6 myobundles per time point). (C) Histogram of myonuclei number per GFP+ myofiber in 3-week myobundles with average and median myonuclei numbers of 7 ± 3.6 and 6, respectively (n = 127 myofibers from 6 myobundles).DOI:http://dx.doi.org/10.7554/eLife.04885.007

Mentions: Myogenic cells were isolated from human muscle biopsies and expanded for 3–5 passages, when they contained a significant fraction of muscle precursors positive for desmin and MyoD (Figure 1—figure supplement 1). Engineered human skeletal muscle ‘myobundles’ were generated using a hydrogel molding technique (Figure 1A, Figure 1—figure supplement 2) we developed for rodent cells (Hinds et al., 2011; Juhas et al., 2014). Following hydrogel compaction for 3–5 days, low serum media was applied to induce myofiber formation and differentiation. After an additional 3–5 days, the myobundles began to spontaneously twitch (Video 1), which was previously reported only in rodent 3D muscle constructs (Dennis and Kosnik, 2000). After 2-week culture, the myobundles contained densely packed and aligned myofibers embedded in a laminin-rich matrix (Figure 1B) and surrounded at the periphery by vimentin+ fibroblasts (Figure 1—figure supplement 3A–C). Mature structure of the myofibers was evident by the expression of myosin heavy chain (MYH), sarcomeric alpha-actinin (SAA) cross-striations, and multiple myogenin+ nuclei (Figure 1C–E and Figure 1—figure supplement 2B–C). Of functional importance, acetylcholine receptors, which are necessary for neuromuscular junction formation, were present at the myofiber surface (Figure 1F). While the majority of expanded myogenic cells fused to form myofibers, a fraction of cells continued to express the satellite cell marker Pax7 (Figure 1G), suggesting regenerative capacity as described in a rat culture model (Juhas et al., 2014). With time in culture, structural maturation of myobundles was evident from the progressive increase in myofiber diameter (13.5 ± 1.5 µm and 21.8 ± 2.8 µm at 1 and 4 weeks of culture, Figure 1H, Figure 1—figure supplement 3D) and expression of the muscle-specific proteins (MYH, SAA, and muscle creatine kinase (MCK), Figure 1I), while myofiber length and myonuclei number (524 ± 70 and 7 ± 3.6, respectively, at 3 weeks of differentiation) remained relatively steady with time of culture (Figure 1—figure supplement 4).10.7554/eLife.04885.003Figure 1.Structure and cellular composition of myobundles.


Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs.

Madden L, Juhas M, Kraus WE, Truskey GA, Bursac N - Elife (2015)

Characterization of myofiber length and myonuclei number.(A) Representative composite image of a 3-week differentiated myobundle formed using 5% GFP expressing myogenic cells to visualize individual myofibers. Scale bar = 500 µm. (B) The average length of GFP+ myofibers as a function of differentiation time (20–50 myofibers per bundle, n = 4–6 myobundles per time point). (C) Histogram of myonuclei number per GFP+ myofiber in 3-week myobundles with average and median myonuclei numbers of 7 ± 3.6 and 6, respectively (n = 127 myofibers from 6 myobundles).DOI:http://dx.doi.org/10.7554/eLife.04885.007
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Related In: Results  -  Collection

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fig1s4: Characterization of myofiber length and myonuclei number.(A) Representative composite image of a 3-week differentiated myobundle formed using 5% GFP expressing myogenic cells to visualize individual myofibers. Scale bar = 500 µm. (B) The average length of GFP+ myofibers as a function of differentiation time (20–50 myofibers per bundle, n = 4–6 myobundles per time point). (C) Histogram of myonuclei number per GFP+ myofiber in 3-week myobundles with average and median myonuclei numbers of 7 ± 3.6 and 6, respectively (n = 127 myofibers from 6 myobundles).DOI:http://dx.doi.org/10.7554/eLife.04885.007
Mentions: Myogenic cells were isolated from human muscle biopsies and expanded for 3–5 passages, when they contained a significant fraction of muscle precursors positive for desmin and MyoD (Figure 1—figure supplement 1). Engineered human skeletal muscle ‘myobundles’ were generated using a hydrogel molding technique (Figure 1A, Figure 1—figure supplement 2) we developed for rodent cells (Hinds et al., 2011; Juhas et al., 2014). Following hydrogel compaction for 3–5 days, low serum media was applied to induce myofiber formation and differentiation. After an additional 3–5 days, the myobundles began to spontaneously twitch (Video 1), which was previously reported only in rodent 3D muscle constructs (Dennis and Kosnik, 2000). After 2-week culture, the myobundles contained densely packed and aligned myofibers embedded in a laminin-rich matrix (Figure 1B) and surrounded at the periphery by vimentin+ fibroblasts (Figure 1—figure supplement 3A–C). Mature structure of the myofibers was evident by the expression of myosin heavy chain (MYH), sarcomeric alpha-actinin (SAA) cross-striations, and multiple myogenin+ nuclei (Figure 1C–E and Figure 1—figure supplement 2B–C). Of functional importance, acetylcholine receptors, which are necessary for neuromuscular junction formation, were present at the myofiber surface (Figure 1F). While the majority of expanded myogenic cells fused to form myofibers, a fraction of cells continued to express the satellite cell marker Pax7 (Figure 1G), suggesting regenerative capacity as described in a rat culture model (Juhas et al., 2014). With time in culture, structural maturation of myobundles was evident from the progressive increase in myofiber diameter (13.5 ± 1.5 µm and 21.8 ± 2.8 µm at 1 and 4 weeks of culture, Figure 1H, Figure 1—figure supplement 3D) and expression of the muscle-specific proteins (MYH, SAA, and muscle creatine kinase (MCK), Figure 1I), while myofiber length and myonuclei number (524 ± 70 and 7 ± 3.6, respectively, at 3 weeks of differentiation) remained relatively steady with time of culture (Figure 1—figure supplement 4).10.7554/eLife.04885.003Figure 1.Structure and cellular composition of myobundles.

Bottom Line: During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength.In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes.Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, United States.

ABSTRACT
Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7(+) cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

Show MeSH
Related in: MedlinePlus