Browsing by Author "Venter, Colin."

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Developing in vitro multi co-culture models and analysis tools for muscle regeneration.
(2019) Venter, Colin.; Niesler, Carola Ulrike.
Skeletal muscle regeneration represents a complex process mediated by non-myogenic cell types. These cells, such as macrophages and fibroblasts, display a range of interactions with muscle stem cells (myoblasts) during myogenesis (the differentiation and fusion of myoblasts into muscle fibres). Our knowledge of these interactions has been elucidated using in vivo and in vitro skeletal muscle models. Although in vivo models are more physiologically relevant, in vitro models, such as co-culture, offer a simpler and cost-effective means to study muscle regeneration. We therefore developed a novel and inexpensive co-culture method using three different cells types, which closely resembled the in vivo microenvironment by permitting a range of cellular interactions. Once this method was established, cellular behaviour in response to various experimental conditions could be evaluated. A second challenge we encountered was that the strategies available to us for assessing myogenesis in vitro were suboptimal in terms of speed and accuracy. We therefore sought to optimize image processing methods to rapidly and accurately quantify cellular numbers (proliferation), wound area (migration) and orientation (alignment) in our co-culture model. We then used these methods to evaluate the roles of macrophages and/or fibroblasts during the early (proliferation and migration) and late (alignment and fusion) stages of myogenesis. We observed a significant increase in myoblast proliferation and migration in response to coculture with either unstimulated macrophages or fibroblasts. In triple co-culture, macrophages continued to promote myoblast proliferation in the presence of fibroblasts. However, the presence of macrophages abrogated the positive effect of fibroblasts on myoblast migration; qualitative analysis also suggested a decrease in fibroblast number. Following analysis of later differentiation, we found that macrophages significantly promoted alignment, but prevented fusion, in a cell density-dependent manner. Fibroblasts, on the other hand, had no significant effect on myoblast alignment, but either promoted (at low fibroblast numbers) or inhibited (at higher fibroblast numbers) fusion. In triple co-culture, the effect of macrophages on myoblast alignment and fusion was unaltered by the additional presence of fibroblasts. In order to determine whether pro-macrophages have a direct quantitative effect on fibroblast number, M1 macrophages were generated following incubation with LPS and then cocultured with a fibroblast population. The latter population was characterised as containing both fibroblasts and their differentiated counterpart, myofibroblasts. A significant decrease in the size of this population (potentially as a result of cell death) was observed in response to M1 macrophages; this decrease was prevented by the addition of LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Subsequent analysis demonstrated that LY294002 decreases macrophage numbers, suggesting a potential mechanism for the rescue of the fibroblast population by this inhibitor. Dexamethasone, on the other hand, caused the fibroblast population to acquire a rounder myofibroblast morphology, but the implications of this morphological change requires further investigation. In this thesis, we presented optimized and novel methods which were used to study skeletal muscle regeneration in vitro. The findings provided new insights into the temporal regulation of myogenesis by non-myogenic cells. During the early stages of myogenesis, macrophages need to increase in number to promote myoblast proliferation, but subsequently resolve with an increase in fibroblast numbers to promote myoblast migration into the wound. During the later stages of myogenesis, macrophage and fibroblast numbers need to subside to promote myoblast alignment and fusion, respectively. The communication between these nonmyogenic cells and the phenotypes they acquire can also indirectly influence myogenesis. The fibroblast population is important for promoting myoblast fusion, but macrophages with an M1 phenotype resulted in death of myofibroblasts. This makes it imperative that the population of M1 macrophages timeously subsides. However, M1 macrophage-mediated death of myofibroblasts was prevented by inhibition of the PI3K pathway which resulted in macrophage, but not myofibroblast, death. This suggests a potential therapeutic target for the treatment of muscle diseases, such as myositis, caused by the dysregulated presence of macrophages.