Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Larry Stokes - University of Mississippi
Co-Author(s): Dr. Manu Platt, Georgia Institute of Technology, Atlanta, GA; Dr. Adeola Michael, Georgia Institute of Technology, Atlanta, GA
Muscle stem cells have the potential to revolutionize therapies to treat degenerative muscular diseases, primarily muscular dystrophy. These versatile cells have a variety of applications ranging from modeling diseases to making engineered living systems. In order to develop cell therapies using muscle stem cells, it is imperative to understand how myogenesis impacts their biological processes, specifically protease modulation. Previous research has shown that muscle stem cells experience an increase in protease levels, in particular cathepsin L, during differentiation at high confluency. Muscle stem cells can experience changes in protease production and activity in response to biochemical and biomechanical stimuli. Here, we examined the effects of protease inhibition on C2C12s, a murine muscle stem cell line, in order to counteract the increase in cathepsin levels. We hypothesized that protease inhibition would decrease cathepsin protease levels thus affecting cell viability. To test this hypothesis, we treated C2C12s with E64, a broad range cysteine cathepsin inhibitor, for 6 and 24 hours. After incubation, the cells were harvested to obtain protein content for analysis using cathepsin zymography and western blotting. Whole cells were harvested after E64 treatment for single cell analysis using the Amnis ImageStream to assess cell viability. Our results showed a decrease in protease activity over time after inhibition. We observed no changes in cathepsin K protein levels, however, cathepsin L protein levels significantly increased after prolonged exposure to E64. After a 24 hour E64 incubation in culture media, cell viability decreased from 74% to 52.2% of the cell population. We postulated that cells under stress will produce more inflammatory cytokines, such as TNF-?, thus affecting cell viability. After a 24 hour incubation with TNF-? to induce cell stress, we observed more cell death compared to control samples incubated in untreated culture media. Future work is aimed at investigating the secretion of proteases into the media as a result of inhibition and cell stress as well as determine a method of assessing cathepsin levels without damaging the cells. By understanding protease biology in muscle stem cells after E64 inhibition, we can better develop a method to control elevated cathepsin protease levels in large batches of differentiating stem cells. In essence, upscaling the production of muscle stem cells will allow for better manufacturing of cell therapies and provides a reliable parameter to gauge the quality of muscle stem cells.
Funder Acknowledgement(s): NSF
Faculty Advisor: Dr. Manu Platt, firstname.lastname@example.org
Role: I was responsible for culturing the cell line, administering the inhibitor and cytokine treatments, conducting the protein analysis by western blotting and zymography, and conducting single cell analysis using the Amnis ImageStream.