Essential environmental cues from the satellite cell niche unraveled

Tissue engineering of skeletal muscle can be used for numerous purposes. The most obvious purposes lie in the field of regenerative medicine: treatment of muscular dystrophies or reconstruction surgery after trauma. In addition, tissue engineered skeletal muscle tissue can be used as a model system to test new drugs or as a model for pressure ulcer research or muscle physiology. Less obvious is its use in the field of consumption as a meat replacement. Contemporary meat production is a heavy burden for the environment, because of an increasing demand of meat. It results in inefficient use of land and water, high emission of greenhouse gasses and risk of spreading of infectious diseases. On top of this, animals often live pitiful lives in bioindustry. Through large scale, industrial production of tissue engineered meat, some of these problems could be diminished. To accomplish this kind of meat production, a number of requirements need to be met. First of all, a cell source is needed that is able to undergo many population doublings, thus produce much progeny, which retains the capacity to differentiate into skeletal muscle. Second, these cells will need to be exposed to the right signals in a three dimensional (3D) environment in order to enable differentiation into mature skeletal muscle tissue. Skeletal muscle cells themselves cannot be used as a cell source, since these cells are built up of many fused cells and are post-mitotic. We therefore make use of the skeletal muscle’s endogenous stem cell population: satellite cells. Satellite cells are responsible for the remarkable regenerative capacity of skeletal muscle tissue; they can repair and regenerate large defects after injury and can respond to changes in load leading to hypertrophy. Unfortunately, satellite cells seem to lose much of their stem cell capacities when cultured in vitro, mostly resulting in a loss of proliferative ability caused by early differentiation. We hypothesized this phenomenon to be caused by loss of the specific environment that these cell usually find themselves in: the niche. Several niche factors can play a role in the satellite cell functioning: growth factors, neighboring cells, extracellular matrix (ECM) proteins, electrical signals from nerves, stretch caused by movement and growth and the elasticity of the environment. In this thesis we investigated the effects of several of these niche factors separately or combined on the proliferation and differentiation capacity of murine satellite cells. We have shown that the choice of ECM protein coating is crucial for all aspects of satellite cell functioning (proliferation, differentiation and maturation). We found maturation (characterized by the presence of cross-striations and spontaneous contractions) to be best on laminin-coated substrates. This seems logical, since the laminin network is the first part of the basement membrane connected to the satellite cells. The elasticity of the matrix influenced both proliferation and maturation of the cells. Proliferation was found to be highest on substrates with an elasticity close to in vivo elasticity of skeletal muscle and classic culturing substrates. For maturation into cross-striated myotubes, it was essential that the elasticity of the substrate was higher than a certain threshold value. Concerning electrical stimulation, we observed an advance in maturation, demonstrated by earlier presence of cross-striations and an upregulation in skeletal muscle differentiation markers. Moreover, electrical stimulation caused a switch in myosin isotype, establishing the possibility to tune the type of skeletal muscle tissue formed (fast or slow type) by electrical stimulation. In contrast, the stretching regime we used had negative effects on muscle maturation, demonstrated by a delay in the development of cross-striations and a downregulation of skeletal muscle differentiation markers. In addition, culturing in different systems has taught us that mere culturing in a 3D environment is much more beneficial for maturation than 2D culturing systems. In conclusion, we have shown that several niche factors play an important role in satellite cell functioning. The results presented in this thesis have important implications for the development of a culturing system for tissue engineered meat.