In vitro skeletal muscle model development through co-culture of cells

Skeletal muscle defects occur because of several reasons and as a promising interdisciplinary approach, skeletal muscle tissue engineering aims the reconstruction of this loss. Transfer of an adjacent muscle tissue in a pediculated form or a distant one as a free flap is a method often applied in treatment of muscular defects and losses but there are limitations for autologous grafting because of the difficulty in procuring donor tissue. Generating new muscle tissue from autologous precursor cells (stem cells) attempts to address this problem via skeletal muscle tissue engineering. The most preferred source of cells for skeletal muscle tissue engineering applications is either a myoblast cell line or the primary satellite cells that can easily be obtained by muscle biopsies and cultured in vitro. Transplanted satellite cells can take part in muscle regeneration and contribute to the satellite cell population in host animals. This makes satellite cells perfect candidates for cell based transplantation therapy for muscle degeneration. Incapability of self-renewal, defective migration of donor cells after transplantation, host immune defense and poor survival are the factors that have affected therapeutic use of satellite cells in clinics. Satellite cells are characterized as a heterogeneous population of progenitors and stem cells. More comprehensive understanding on satellite cell hierarchy is needed at single cell level and their performance should be followed over time. Skeletal muscle that contains in vivo functionality requires the induction of main types of tissues such as vascular, nervous, muscle and connective tissues. Henceforth, numerous connective tissues cover the different muscle bundle, the muscle fibers and the whole muscle, while a skeletal muscle is instilled by capillaries and joined to the nerve branches. Vascularization is an important concern in capacious tissues having high oxygen demand such as the skeletal muscle. It is responsible for the storage and breakdown of glucose and fat since it is a highly metabolic organ. On top of that, skeletal muscle is the primary tissue accountable for the augmented glucose metabolism during exercise and hyperinsulinemia. It is a challange to mimic this complex system in vitro conditions. Myofiber is large single cell which is formed by fusion of many cells during development. Bundling of these muscle fibers leads to muscle formation. Skeletal muscle cells are innervated by motor neurons. Each neuron can control a small number of myofibers and fires a contraction. There is a need for neuronal stimulation during myogenesis. With the help of tissue engineering principles, differentiated skeletal muscle tissue can be restored by utilizing multiple stem or progenitor cells from different sources. Self regeneration of mature muscle fibers that are vascularized and multinucleated is not possible because of terminal differentiation. Satellite cells or myoblasts are capable of maintaining the regenerative potential of injured muscle tissue. As it is common in other tissues, the vascular network in skeletal muscle contains arteries splitting into increasingly smaller vessels. In skeletal muscle, a terminal arteriole gives rise to collections of capillaries that run parallel to muscle fibers, and each muscle fiber can be supplied by numerous unalike groups of capillaries from independent terminal arterioles. As the other invaluable component of the muscle tissue, in vitro establishment of innervation by the motor neurons would better mimic the native tissue and provide an opportunity to test the effect of drugs targeting the neuromuscular junctions. Both innervated and vascularized human muscle tissue construct is expected to have a reduced time for functional integration in the body and eliminate the species-related variability associated with animal-based systems when used in drug discovery and toxicology studies. This study presents a report on the generation of a novel human muscle tissue model using skeletal muscle derived stem cells and human umbilical vein endothelial cells (HUVECs). In this study, isolation of skeletal muscle stem cells was carried out by pre-plate method. They were differentiated into neuron-like cells that were negative for the astrocyte marker glial fibrillary acidic protein (GFAP) and positive for neuronal beta-tubulin III and nestin. Satellite cells matured in vitro into myoblasts and fused with each other and create the myotubes. Capillary network and multinucleated myotube formation were provided by the co-culture of skeletal muscle stem cells with the HUVECs under optimized fetal bovine serum and media conditions. Cells were immunostained for the myogenic marker desmin and the endothelial marker CD31. It was observed that HUVECs did not survive in a DMEM/F12 medium, supplemented with 5%, 10% and 20% FBS. When EGM-2 medium was mixed with DMEM/F12, however, the HUVECs survived and proliferated The best media combination for the formation of both myotubes and capillary networks was found as DMEM/F12 (10% FBS):EGM-2 (2% FBS) (1:1) mixture. The seeding of the neuron-like cells derived from the human skeletal muscle stem cells was performed on the vascularized myotubes to form neuromuscular junctions. It was found that the neuron-like cells were in contact with the myotubes after 24 hours of co-culture. Real time observation with time lapse imaging used in order to investigate neural differentiation of human skeletal muscle derived stem cells (hSkMSCs) and the behavior of cells in co-culture conditions. Finaly, electrophysiological study was carried out and contraction of the multinucleated cells was observed under the phase contrast microscope. This study presents a novel in vitro human skeletal muscle model with advanced capillary networks with an interaction of myotubes and neurons, and it can be utilized in the in vitro testing of drugs and also used for the regeneration of skeletal muscles by means of cellular therapy or cell-laden tissue engineered muscle constructs.