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

The influence of serum-free culture conditions on skeletal muscle differentiation in a tissue-engineered model

The influence of differentiation medium (DM) components on C2C12 murine myoblast differentiation has only been studied in monolayer cultures. Serum-free formulations have been applied that omit the use of sera with unknown composition. The goal of the present study was to compare the influence of serum-free media on C2C12 differentiation in 3-dimensional tissue-engineered muscle constructs. Myoblast proliferation and differentiation in media containing Ultroser G (DMU), insulin-like growth factor (IGF)-I (DMI), or both (DMUI) were compared with those induced by more-traditional media containing horse serum (HS) or horse serum and IGF-I (HSI). Effects of the applied media were assessed from gross construct morphology, total protein content, creatine kinase activity, and tissue viability. Addition of IGF-I (HSI) to the standard DM (HS) improved myoblast differentiation in muscle constructs. Even better results were obtained using DMU and DMUI culture conditions. DMI could not induce differentiation or maintain cell viability. Serum-free culture medium supplemented with DMU or DMUI accelerates and improves myoblast differentiation in engineered muscle tissue better than the gold standard HS

Tuning myosin heavy chain isotype expression by electrical stimulation

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Essential environmental cues from the satellite cell niche: Optimizing proliferation and differentiation

The use of muscle progenitor cells (MPCs) for regenerative medicine has been severely compromised by their decreased proliferative and differentiative capacity after being cultured in vitro. We hypothesized the loss of pivotal niche factors to be the cause. Therefore, we investigated the proliferative and differentiative response of passage 0 murine MPCs to varying substrate elasticities and protein coatings and found that proliferation was influenced only by elasticity, whereas differentiation was influenced by both elasticity and protein coating. A stiffness of 21 kPa optimally increased the proliferation of MPCs. Regarding differentiation, we demonstrated that fusion of MPCs into myotubes takes place regardless of elasticity. However, ongoing maturation with cross-striations and contractions occurred only on elasticities higher than 3 kPa. Furthermore, maturation was fastest on poly-D-lysine and laminin coatings. Copyrigh

Urine flow cytometry can rule out urinary tract infection, but cannot identify bacterial morphologies correctly

The diagnosis of urinary tract infection (UTI) by urine culture is a time-consuming and costly procedure. Usage of a screening method, to identify negative samples, would therefore affect time-to-diagnosis and laboratory cost positively. Urine flow cytometers are able to identify particles in urine. Together with the introduction of a cut-off value, which determines if a urine sample is subsequently cultured or not, the number of cultures can be reduced, while maintaining a low level of false negatives and a high negative predictive value. Recently, Sysmex developed additional software for their urine flow cytometers. Besides measuring the number of bacteria present in urine, information is given on bacterial morphology, which may guide the physician in the choice of antibiotic. In this study, we evaluated this software update. The UF1000i classifies bacteria into two categories: ‘rods’ and ‘cocci/mixed’. Compared to the actual morphology of the bacterial pathogen found, the ‘rods’ category scores reasonably well with 91% chance of classifying rod-shaped bacteria correctly. The ‘cocci/mixed’ category underperforms, with only 29% of spherical-shaped bacteria (cocci) classified as such. In its current version, the bacterial morphology software does not classify bacteria, according to their morphology, well enough to be of clinical use in this study populatio