Abstract:
The experiment conducted in this research article explored the possibilities of using muscle stem cells to repair damaged and dysfunctional muscles. This type of therapy could be a form of treatment for people with muscular dystrophy. The disease that was focused on here was Duchenne muscular dystrophy (DMD). To understand the aim of this research, we first need to understand Duchenne muscular dystrophy. DMD is a severe type of muscular dystrophy, one of many in a group of genetic disorders characterized by progressive muscle degeneration and weakness. Because it is caused by mutations in the DMD gene, which is located on the X chromosome, it primarily affects boys. (DMD is inherited in an X-linked recessive manner. (Since males have only one X chromosome (XY), a single mutated copy of the gene will cause the disease. Females have two X chromosomes (XX), so they are typically carriers if only one X chromosome carries the mutation. Female carriers usually do not exhibit symptoms but can pass the mutated gene to their offspring.) Duchenne muscular dystrophy affects approximately 1 in every 5000 male births. The DMD gene encodes for a protein called dystrophin which is essential for maintaining the structural integrity of muscle cells. Dystrophin acts like a shock absorber, helping to protect muscle fibers from damage during contraction and relaxation. In the absence of functional dystrophin, muscle cells become fragile and easily damaged. This leads to a variety of problems. Repeated cycles of damage and repair lead to muscle fibers being replaced by scar tissue (fibrosis) and fat, rather than new muscle cells regenerating. This muscle damage triggers chronic inflammation, worsening tissue damage. Over time, the cumulative damage results in the gradual weakening of the muscles. This affects the whole body, especially the heart and respiratory muscles. Weakness of the respiratory muscles leads to breathing difficulties and heart muscle weakness (cardiomyopathy) develops, leading to heart failure. The life expectancy of patients with DMD is only 18-25 years.
Extracellular vesicles:
Another important thing to understand. Extracellular vesicles (EVs) are membrane-bound particles released by cells into the extracellular environment. They play a crucial role in cell-to-cell communications and are involved in various physiological and pathological processes by transporting molecules such as proteins, lipids, and nucleic acids. This article uses EVs derived from muscle stem cells (MuSC-Evs). Muscle stem cells release various types of Evs, including exosomes (30-150 nm) and microvesicles (100-1000 nm). Exosomes are involved in intercellular communication, immune responses, and the transfer of genetic material and proteins between cells. On the other hand, microvesicles participate in various other processes which include inflammation and cellular signaling. MuSC-Evs mediate communications between muscle stem cells and other cell types within the muscle tissue. They control the repair and regeneration process.
Activation of MuSCs:
Muscle regeneration is a complex process involving the activation of MuSCs, proliferation, and how they differentiate into myoblasts, which fuse into the preexisting myotubes to facilitate regeneration. When a muscle is injured, whether it’s from exercise, disease, etc. damaged muscle fibers release signals for MuSC activation. Myoblasts come from MuSCs and upon activation, they begin proliferating to increase their numbers. As they exit the cell cycle, they differentiate into specialized muscle cells with designated functions called myocytes. The myoblasts then fuse to form myotubes. A myotube is a type of cell that will develop into a mature muscle fiber. To put it simply, in response to muscle injury or stress, MuSCs activate, proliferate, and differentiate into myoblasts. These myoblasts then fuse with damaged muscle fibers to form new fibers, contributing to the process of muscle repair and regeneration.
Experiment:
All the muscles of the lower limb were removed from both limbs of the 6 mice used for this part of the experiment. The muscles were then minced into a slurry and the minced tissue was transferred to wash. It was then placed into an ultracentrifuge and spun at 500x g, which converts over to about 40,000 rpm. The force that the minced tissue was being spun at separates the larger molecules from the smaller molecules due to their different densities. The MuSC could then be isolated from the rest of the slurry. The MuSCs were then taken and grown in a cell culture for six days in a 37 C incubator. Then C2C12 myoblasts, a type of muscle cell line, were cultured so they could reach a fully differentiated state into the myotubes. The C2C12 myoblasts were seeded at a density of 1.0 × 10^4 cells per cm². This means for every square centimeter of the culture vessel, 10,000 cells were initially placed. The cells were grown in an incubator set at 37°C with 5% carbon dioxide (CO2). These conditions provide an optimal environment for the cells to grow, mimicking the physiological conditions of the human body. The myoblasts were allowed to proliferate (multiply) until they reached 80-90% confluency. Confluency refers to the percentage of the culture vessel surface covered by cells. At this stage, the cells are densely packed but not yet overgrown, which is optimal for inducing differentiation. Once the cells reached the desired confluency, the growth media was replaced with differentiation media to induce the myoblasts to become myotubes. The myoblasts were maintained in differentiation media for 5 days. During this time, the cells undergo morphological and biochemical changes to become myotubes. The fully differentiated myotubes resemble the structure and function of mature muscle fibers. Only the fully differentiated myotubes were used throughout this experiment. The Nanosight NS300 was used for this analysis. The instrument is equipped with a 532-nm green laser, which is used to illuminate the EVs in the sample for visualization and measurement. Three videos were recorded for one minute each to ensure sufficient data collection and accuracy. EV samples were prepared with a 1:140 dilution. This means one part of the EV sample was mixed with 139 parts of sterile, filtered phosphate-buffered saline (PBS) to achieve a total volume of 1 mL. Phosphate-buffered saline is used to maintain the pH and osmolarity of the solution, ensuring the stability of the EVs during analysis. The next part of the article details the experimental procedures for isolating, labeling, and analyzing the uptake of MuSC-EVs by the C2C12 myotubes. A total of 1.8 × 10^11 MuSC-EVs were labeled using fluorescent dye. The EVs were then incubated with the dye for 2 hours at 37°C. 50 μg (1 millionth of a gram) of labeled MuSC-EVs were added to the media of C2C12 myotubes. The myotubes were incubated with the EVs at 37°C for 24 hours. After incubating for 24 hours, the myotubes were washed to remove any unincorporated labeled proteins. Imaging of the MuSC-EV protein uptake was performed using a confocal microscope system, which is a specialized form of standard fluorescence microscopy. Media was collected from cultured MuSCs 24 hours after plating. EVs were isolated from the collected media and the isolated EVs were characterized using nanoparticle tracking analysis (NTA). The number of EVs released per MuSC over 24 hours was calculated using data derived from NTA and the seeding density of the MuSCs. Each MuSC released approximately 2.35 × 10^5 ± 3.10 × 10^4 EVs over the 24 hours. The mean size of MuSC-EVs was found to be 125.7 ± 1.7 nm. These findings indicate that MuSCs primarily release EVs in the size range of small and medium-sized EVs. Now that they have demonstrated that MuSCs can release large amounts of EVs, they started the next part of their research which was to demonstrate that muscle stem cell-derived extracellular vesicles can deliver proteins to recipient cells, specifically differentiated C2C12 myotubes, which act as adult muscle fibers in vitro. (“In vitro” means done in a laboratory dish or a test tube while “in vivo” means done on a living organism. If an experiment passes the in vitro phase, the next step would be to do one in vivo.) Differentiated C2C12 myotubes were used as the recipient cells and the labeled MuSC-EVs were added directly to the culture media of the myotubes. The myotubes were incubated with the labeled MuSC-EVs for 24 hours. After the 24-hour incubation period, the myotubes were visually inspected using fluorescent microscopy. There appeared to be an abundance of fluorescent puncta within the cultured myotubes, suggesting that the cells were able to uptake the CFSE-labeled proteins from the MuSC-EVs. All experimental groups were compared to untreated control myotubes to ensure that the observed effects were due to the MuSC-EVs. The conclusions from this part suggested that MuSCs release a substantial amount of EVs and these EVs can deliver proteins to differentiated C2C12 myotubes. Although significant protein uptake occurs as early as 2 hours, peaks at 24 hours, and is sustained up to 48 hours, suggesting stable incorporation and resistance to rapid degradation.
Conclusion, Limitations, and Final Thoughts:
The primary therapeutic function of MuSCs is their ability to differentiate into myoblasts and fuse with damaged muscle fibers, aiding in muscle regeneration. However, a significant number of MuSCs are needed to treat systemic muscle diseases. In a later experiment done on mice with DMD, trillions of MuSCs were needed for one mouse. And during ex vivo expansion (growth outside the organism), MuSCs tend to lose their ability to form muscle tissue (myogenic potential). Other limitations that the researchers could have encountered during their research may be the co-isolation of non-EV components such as proteins, lipoproteins, and other cellular debris, which can occur and complicate downstream analyses. Other factors such as the EV’s low yield could also cause erroneous results. As they tried to scale up the production to meet their experimental needs, the process would have been challenging and may have required optimized culture conditions and bioreactor systems. Lack of access to special equipment may negatively affect the purity of the MuSC-EVs collected. Furthermore, the mechanisms by which MuSC-EVs exert their effects are not fully understood, complicating the interpretation of experimental results. Ensuring the bioactivity and functional integrity of MuSC-EVs after isolation and storage can also be difficult, as EVs may lose functionality over time or under suboptimal storage conditions. The lack of standardized reporting guidelines for EV research can lead to inconsistencies in data interpretation and comparison across studies. However, this study also showed certain advantages and the potential of MuSC-Evs. For example, cells are able to continuously release EVs, making them potentially more efficient and scalable for therapeutic use compared to whole-cell therapies. Other advantages include MuSC-EVs have been shown to reverse mitochondrial dysfunction caused by oxidative stress (Oxidative stress can damage cells by producing harmful molecules called reactive oxygen species, which can impair mitochondrial function. Oxidative stress plays a significant role in various muscle diseases and disorders.) and the EVs from MuSCs help restore the energetic phenotype of damaged myotubes. This means that MuSC-EVs help the myotubes regain their normal energy production and function, which is very important for muscle health and performance. The therapeutic response observed with MuSC-EVs is also profound, suggesting they could be an effective treatment for muscle damage. The use of EVs is considered simple compared to whole-cell therapies, as they can be more easily produced and administered. The research suggests that MuSC-EVs are a promising therapeutic tool for muscle regeneration and repair. They offer several advantages over traditional cell-based therapies, including simplicity, safety, and scalability. However, many more future studies will be required to help better understand their mechanisms of action and broaden their potential applications in treating various muscle diseases.
10/6/2024
