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Essential environmental cues from the satellite cell niche unraveled
Get PDFTissue 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
Molecular MRI of VCAM-1 expression in atherosclerotic mice using targeted micellular iron oxides
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The interplay between the extracellular environment and cardiomyocyte progenitor cells for material based cardiac repair
Get PDFThe essence of biophysical cues in skeletal muscle tissue engineering
Get PDFSkeletal muscle is an appealing topic for tissue engineering because of its variety in applications. Evidently, tissue engineered skeletal muscle can be used in the field of regenerative medicine to repair muscular defects or dystrophies. Engineered skeletal muscle constructs can also be used as a model system for drug-screening or to study muscle physiology and the etiology of pressure sores. Besides these well known applications, a new field of interest with high societal impact has arisen, being in vitro cultured meat. Contemporary large-scale farming and transportation of livestock brings along a high risk of infectious animal diseases and environmental burden through greenhouse gas emission. In vitro cultured meat can dramatically reduce these risks and improve animal welfare. Although major advances have been made so far in skeletal muscle tissue engineering, the maturation level of the engineered muscle constructs is still not satisfactory. The requirements of the engineered skeletal muscle constructs are similar for both regenerative medicine and in vitro meat production: mature functional skeletal muscle tissue is required that can produce force within the physiological range. To improve the maturation process of skeletal muscle progenitor cells we have focused on mimicking the native environment of these cells. In particular, we have focused on biophysical cues that play an essential role in the regeneration of skeletal muscle tissue in vivo. These cues were investigated in conventional 2D cultures, as well as in 3D model systems that are physiologically more relevant. An important biophysical cue that was investigated is electrical stimulation, since nerve stimulation is known to be a prerequisite for myotube formation in vivo. We have shown that electrical stimulation results in an acceleration of the formation of cross striations and increased expression levels of muscle maturation markers in 2D and 3D experiments. These effects were observed in cultures of the conventional C2C12 cell line and primary muscle progenitor cells (MPCs). More specifically, electrical stimulation of 3D cultures with MPCs resulted in a shift of myosin heavy chain (MHC) expression towards slower isoforms. Electrical stimulation can be implemented in skeletal muscle tissue engineering strategies to improve efficiency of the culture process and to tune MHC expression, which may be relevant for the final texture of the engineered constructs. Mechanical cues also play an important role in muscle development in vivo, both in the embryonic phase and in adults. Stretch can result in hypertrophy of skeletal muscle tissue and can therefore improve texture and force production of tissue engineered skeletal muscle constructs. However, our mechanical stimulation protocol, with applied strains within the physiological range, resulted in impaired muscle maturation in both C2C12 and primary MPCs and is therefore not useful for skeletal muscle tissue engineering. A major finding of the results presented in this thesis is that the 3D environment in which muscle progenitor cells are cultured is essential for myogenesis. Sarcomere formation was faster in a 3D hydrogel based environment, compared to conventional 2D cultures. In 2D, sarcomere formation is optimal on substrates with a stiffness similar to in vivo skeletal muscle tissue, between 3-12 kPa. However, the stiffness of our 3D hydrogel systems was considerably lower than this range and muscle formation still progressed rapidly. We concluded that not the substrate stiffness itself, but the ability of cells to develop tension is essential for the formation of cross striations. Both in 2D and 3D settings we demonstrated that the Rho-associated kinase plays a role in this process, since no cross striations were observed when this kinase was inhibited. Additionally, 3D culture methods that enable an increase in cellular tension result in acceleration of the maturation process. MPCs cultured in 3D resulted in more mature skeletal muscle tissue compared to C2C12 and are therefore the preferred cell source for tissue engineering applications. However, their proliferative capacity remains limited. We showed that the C2C12 cell line, which is readily accessible and easy to culture and differentiate, can be used as a model system to design 3D culture methods and biophysical stimulation regimes. In conclusion, we have shown that several biophysical cues are important for in vitro skeletal muscle maturation. The results presented in this thesis have contributed to the technology that can realize the in vitro production of meat
The essential role of the biophysical environment of cardiac progenitor cells for regenerative therapy
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Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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