Skeletal muscle tissue engineering: A maturation model promoting long-term survival of myotubes, structural development of the excitation–contraction coupling apparatus and neonatal myosin heavy chain expression

Abstract

The use of defined in vitro systems to study the developmental and physiological characteristics of a variety of cell types is increasing, due in large part to their ease of integration with tissue engineering, regenerative medicine, and high-throughput screening applications. In this study, myotubes derived from fetal rat hind limbs were induced to develop several aspects of mature muscle including: sarcomere assembly, development of the excitation-contraction coupling apparatus and myosin heavy chain (MHC) class switching. Utilizing immunocytochemical analysis, anisotropic and isotropic band formation (striations) within the myotubes was established, indicative of sarcomere formation. In addition, clusters of ryanodine receptors were colocalized with dihydropyridine complex proteins which signaled development of the excitation-contraction coupling apparatus and transverse tubule biogenesis. The myotubes also exhibited MHC class switching from embryonic to neonatal MHC. Lastly, the myotubes survived significantly longer in culture (70-90 days) than myotubes from our previously developed system (20-25 days). These results were achieved by modifying the culture timeline as well as the development of a new medium formulation. This defined model system for skeletal muscle maturation supports the goal of developing physiologically relevant muscle constructs for use in tissue engineering and regenerative medicine as well as for high-throughput screening applications.

Local Tissue Geometry Determines Contractile Force Generation of Engineered Muscle Networks

“…S1D). Compared with the initial cell/gel volumes of 55.85, 53.56, and 51.88 mm 3 (for PL = 0.6, 1.2, and 1.8 mm, respectively) injected to fill the tissue molds, the final tissue volumes significantly decreased with the increase in PL by 83.0% -1.1%, 84.2% -1.1%, and 87.1% -0.8%, respectively, showing that the longer posts also yielded a higher degree of cell-mediated gel compaction.…”

“…1,2 Engineered muscle constructs can also be used as three-dimensional (3D) tissue models that complement the conventional two-dimensional (2D) cell cultures and animal models in studying the processes of muscle development, regeneration, and disease. 3,4 While several important studies. [5][6][7] have focused on developing methods to improve survival, neovascularization, and functional integration of engineered muscle tissues in vivo, the force generating capacity of skeletal muscle constructs still remains significantly inferior to that of the native muscle.…”

Neuromuscular Junction Formation Between Human Stem-Cell-Derived Motoneurons and Rat Skeletal Muscle in a Defined System

“…21 This system successfully supported the differentiation of both dissociated skeletal muscle cells (obtained from the hind limb of embryonic rat) and the maturation of human MNs from fetal SCs. The coculture was characterized by morphology, immunocytochemistry, and electrophysiology.…”

A system for studying mechanisms of neuromuscular junction development and maintenance

“…Much attention has been given to the development and improvement of the neuronal compartment with the emergence of embryonic stem cells (Chipman et al, 2014;Das et al, 2007;Soundararajan et al, 2007) and induced pluripotent stem cells (Bohl et al, 2015;Burkhardt et al, 2013;Egawa et al, 2012); however, the muscle counterpart has been underexploited and its potential underestimated. Previous studies failed to produce convincing data on muscle differentiation either because muscle cell lines, such as C2C12, incapable of producing highly differentiated myofibers, were used or because the muscle compartment was overlooked (Arnold et al, 2012;Chipman et al, 2014;Das et al, 2009Das et al, , 2010Umbach et al, 2012). The use of such cell lines might also preclude synaptogenesis and/or synaptic differentiation.…”

“…To date, co-culture methods established from various species have been described, including mouse (Morimoto et al, 2013;Zahavi et al, 2015), rat (Das et al, 2010;Southam et al, 2013), Xenopus (Lu et al, 1996;Peng et al, 2003) and chick (Frank and Fischbach, 1979), and also heterologous co-cultures built from motor neuron and muscle cells obtained from different species, such as rat-human , mouse-human (Son et al, 2011) and mouse-chick (Soundararajan et al, 2007). However, these co-culture methods resulted in the formation of immature myofibers (thin muscle fiber, with centrally localized nuclei and no transversal triads) with immature sarcomeric structures (Das et al, 2007(Das et al, , 2009Southam et al, 2013). Moreover, previous models did not take advantage of their co-culture system to analyze other post-synaptic structures such as the formation of muscle-specific tyrosine kinase (MuSK) and Rapsyn (also known as Rapsn) clusters which are formed as agrin-induced signaling sparks off and which are essential to the formation of acetylcholine receptor (AChR) clusters.…”