Developing in vitro multi co-culture models and analysis tools for muscle regeneration.
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Date
2019
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Abstract
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.
Description
Doctoral Degree. University of KwaZulu-Natal, Pietermaritzburg.