The research contained in this thesis was completed by the candidate while based in the Biochemistry discipline, School of Life Sciences at the College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa. The content of this work has not been submitted in any form to another university and, except where the work of others is acknowledged in the text, the results reported are due to research carried out by the candidate.
PLAGIARISM
A triple co-culture method to investigate the effect of macrophages and fibroblasts on myoblast proliferation and migration. Cellular alignment and fusion: Quantifying the effect of macrophages and fibroblasts on myoblast terminal differentiation. 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.
In triplicate co-culture, macrophages continued to induce myoblast proliferation in the presence of fibroblasts. In triplicate co-culture, the effect of macrophages on myoblast elongation and fusion was unchanged by the additional presence of fibroblasts. This suggests a potential therapeutic target for the treatment of muscle diseases, such as myositis, caused by the dysregulated presence of macrophages.
INTRODUCTION
In the current study, we first aimed to evaluate the effect of macrophages and/or fibroblasts on the behavior of myoblasts in vitro. The final objective was to determine the effects of macrophages and/or fibroblasts on myoblast proliferation, migration, alignment and fusion. Therefore, our second aim was to evaluate the effect of pro-inflammatory (M1) macrophages on the population of fibroblasts/myofibroblasts in a muscle regenerative in vitro microenvironment.
The second aim was then to evaluate the effect of M1 macrophages on the number of fibroblasts/myofibroblasts. This article describes a novel in vitro co-culture method that we used to evaluate the effect of macrophages and/or fibroblasts on myoblast proliferation and migration. This chapter focuses on the characterization of macrophages and (myo)fibroblasts, the effect of M1 macrophages on proto-myofibroblasts, and the mechanism of M1 macrophage-mediated proto-myofibroblast cell death.
MYOGENESIS IN A DISH: A GUIDE TO GENERATING IN VITRO
Regulation of myogenesis has been previously evaluated using various in vivo and in vitro experimental models. This may be due to differences in the in vitro models, such as the cell lines used. In vitro models typically involve the use of co-culture methods (to study paracrine and juxtacrine signaling), conditioned media (to study paracrine signaling) or cell-derived matrices (to study the effect of signaling via deposited matrix factors) [ 50 -52 ].
Co-culture systems represent an in vitro model where two or more different types of cells are grown together in a common environment to study intercellular communication [50]. Similarly, our laboratory previously adapted in vitro fertilization (IVF) culture plates ( Figure 3G ) for co-culture [ 46 ]. Myogenesis can be mimicked in vitro to study the effects of specific experimental conditions on cell behavior such as proliferation, apoptosis, motility, alignment, and fusion.
RAPID QUANTIFICATION OF CELLULAR PROLIFERATION AND
We developed optimized protocols for rapid and accurate quantification of the number of adherent cells and wound area using ImageJ, an open source image processing program. To manually measure the wound surface, the edges of the wound were drawn using the Motic Images Plus 2.0 ML software [15]. The MinError threshold (Image → Adjust → Auto Threshold: . MinError) was then applied to automatically detect the wound area.
If the wound area was not precisely selected, it can be limited manually (Image → Adjust → Threshold). As a result, when the percentage of wound closure was assessed, a significant difference was found between the manual (12.13 ± Figure 2: Wound surface quantification: comparison of manual and automated (ImageJ) identification methods. would depend on the image quality.
A TRIPLE CO-CULTURE METHOD TO INVESTIGATE THE EFFECT
A significant increase in proliferation and migration of myoblasts was observed after co-culture with macrophages or fibroblasts. However, triple co-culture of macrophages, fibroblasts and myoblasts revealed that the presence of macrophages prevented fibroblasts from maintaining this positive effect on myoblast migration. Method Summary: A novel triple co-culture method has been developed to facilitate the co-culture of macrophages, fibroblasts and myoblasts.
The cell lines used by the co-culture method were cultivated in the same culture medium and did not require additional optimization. To initially co-culture the two cell populations, macrophages were placed at the outer edge of the well, while myoblasts were cultured in the center of the well ( Figure 2A,B ). This co-culture method was then successfully repeated using fibroblasts instead of macrophages (Figure 2C).
To establish a “triple co-culture”, macrophages and fibroblasts were cultured on the outer edge of the well, while myoblasts were cultured in the center of the well (Figure 2D). Using our co-culture method, we tried to determine the effect of macrophages and fibroblasts on the proliferation and migration of myoblasts. To determine the combined effect of macrophages and fibroblasts on myoblast proliferation and migration, a triplicate co-culture experiment was performed where myoblasts were co-cultured with 40 x 103 macrophages and 40 x 103 fibroblasts.
Analysis of myoblast proliferation revealed that triple co-culture of myoblasts with macrophages and fibroblasts did not significantly alter myoblast numbers when compared to conditions using macrophages or fibroblasts alone (Figure 4A). However, when myoblast migration was analyzed, triple co-culture of myoblasts with macrophages and fibroblasts abolished the previous significant positive effect of fibroblasts on myoblast migration (Figure 4B). Triplicate co-culture studies using macrophages, fibroblasts and myoblasts revealed that the presence of macrophages negates the pro-migratory effects of fibroblasts on myoblasts.
In vitro studies have shown that increasing fibroblasts in co-culture also promotes myoblast migration [18] and that fibroblasts protect myoblasts from apoptosis during differentiation, which promotes myotube formation [36].
CELLULAR ALIGNMENT AND FUSION: QUANTIFYING THE
Finally, triplicate co-culture revealed that the effect of macrophages on myoblast alignment and fusion was unaltered by the additional presence of fibroblasts. Damage to skeletal muscle leads to the disruption of myofibers and the extracellular matrix (ECM) that surrounds them [2]. Once established, we then apply this protocol to determine the effect of macrophages and fibroblasts on the alignment of myoblasts during fusion.
To determine the effect of an inflammatory and fibrotic environment on myoblast differentiation, triple co-cultures of macrophages and fibroblasts with myoblasts were performed (Figure 6). Next, we sought to use our established method to determine the effect of macrophages and fibroblasts on myoblast alignment and fusion. Finally, the presence of fibroblasts did not alter the effect of macrophages on myoblast differentiation.
In our previous studies, we evaluated the direct role of macrophages and fibroblasts on myogenesis; in the current study we sought to better understand the intercellular communication between macrophage and fibroblast populations. In contrast, dexamethasone increased the circularity of the myofibroblast population, indicating a decrease in the number of residual fibroblasts within the population. Fibroblasts have been shown to differentiate into myofibroblasts in the presence of serum and/or TGF-β; Differentiation is thought to be reversible [27-29].
Macrophages were then co-cultured with myofibroblasts or myoblasts in the presence of LPS to determine the effect of a pro-inflammatory environment on myofibroblast and myoblast proliferation (Figure 3). Furthermore, myoblasts cultured in the presence of M1 macrophages (Mϕ+LPS+) were present with a relative cell number of 297. Myoblasts maintained in the presence of LPS (Mϕ-LPS+) and were present with relative cell numbers of 145 ± 6%, which did not differ significantly from the control.
The mechanism by which M1 macrophages reduce myofibroblast numbers was assessed by co-culturing macrophages and myofibroblasts in the presence of LPS and 2% serum in the presence or absence of soluble inhibitors (Figure 4). The effect of the inhibitor LY294002 and glucocorticosteroid dexamethasone on myofibroblast morphology in the presence of macrophages and LPS was evaluated (Figure 5). Dexamethasone continued to significantly increase the mean circularity of myofibroblasts from DEX-LPS+) to DEX+LPS+) in the presence of LPS.
CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER
We mainly investigated the effect of the presence of macrophages and/or fibroblasts on myoblast behavior, but not the mechanism of communication. Take images of the cells (3-5 fields of view per repetition for 2-3 repetitions) with a camera-attached bright-field microscope (4x objective lens). Take images of the wound area at different time points (0, 3, 5 and 7 hours) with a camera-attached microscope (4x objective lens).
APPENDIX B: PROTOCOL FOR: A TRIPLE CO-CULTURE METHOD TO INVESTIGATE THE EFFECT OF MACROPHAGES AND FIBROBLASTS ON MYOBLAST PROLIFERATION AND MIGRATION. To evaluate the relationship between these cell types, we first developed a new, inexpensive in vitro co-culture method for two or more cell types with some cell contact; we evaluated the effect of macrophages and/or fibroblasts on myoblast proliferation and migration. We found that macrophages and fibroblasts can promote both myoblast proliferation and migration, depending on their cell numbers; triple co-culture of macrophages, fibroblasts and myoblasts revealed that macrophages and fibroblasts continued to promote myoblast proliferation, but the presence of macrophages impaired the positive effect of fibroblasts on myoblast migration.
We then developed a method to rapidly determine cell orientation and quantify alignment and used our co-culture technique to determine the effect of macrophages and/or fibroblasts on myoblast alignment and fusion. Triplicate co-culture of the cells showed that the effect of macrophages on myoblast alignment remains unchanged in the presence of fibroblasts. Proliferation and migration of myoblasts in the center of the well were then analyzed as described below.
To analyze the effect of macrophages on fibroblast phenotype, macrophages (40 x 103) were plated in growth medium at the edge of the well after which fibroblasts (40 x 103) were allowed to attach in the center of the well. Triplicate co-culture studies using macrophages, fibroblasts and myoblasts revealed that the presence of macrophages negated the promigratory effects of fibroblasts on myoblasts. Previous studies using more than two cell types in co-culture are limited (31); in fact, no previous studies have been performed to determine the combined effect of macrophages and fibroblasts on myoblast proliferation and migration.
Finally, triple co-culture revealed that the effect of macrophages on myoblast alignment and fusion remains unchanged by the additional presence of fibroblasts. Co-culture of macrophages and/or fibroblasts with myoblasts was established as described by Venter and Niesler[6]. Finally, additional triple co-culture studies showed that this effect of macrophages on myoblast differentiation was not altered by the presence of fibroblasts.
