In 2006, a Japanese researcher named Yamanaka discovered a chemical concoction which would allow the dedifferentiation of fated cells to once again become induced pluripotent stem cells (iPSCs). This discovery had significant impact in the world of regenerative tissue engineering, as it allowed for the indefinite creation and proliferation of complex cell types cultured from the patient’s own cells. In particular, the Bursac lab under which I am currently researching studies how iPSCs can be used to specify functional synthetic myocytes, both skeletal and cardiac. However, the large roadblock in tissue engineering research currently is that tissue fated from iPSCS are not able to generate as much force and muscular volume as primary myocytes.
The projects I am working on seek to better understand the roles that vascularization and innervation play in the development of functional skeletal muscle tissue, and more importantly, how co-culturing endothelial cells or neurons can allow muscles to exhibit greater forces. As one may expect, working with cell cultures are very demanding and requires a comprehensive set of procedures to ensure the growth and differentiation of each tissue type, as well as the necessary processing in order to understand the capabilities or functionality of the cells.
Although I’m only one week in, I’ve definitely picked up a plethora of laboratory techniques which were necessary in order to cover all aspects of an experiment. I learned how to create 2D and 3D growth media which facilitates the growth of cells within the first week of culture. After this, the cells will spend 2-3 weeks in a differentiation media which causes them to fuse and interact with each other and form tissue. It is during this step where endothelial/neuron cells will interact closely with the formed myofibers and form supporting vasculature or neuromuscular junctions. To understand the state of the muscle fibers at different points of the experiment, the tissue can be used in many ways. One of the most important of course is force testing, in which we rig bundles to a sensitive force guage and electrically stimulate them to understand their impulse responses or tetanus response. Another common way to understand the bundles is to do cross section stains, which requires the use of the cryostat machine I described last week, or whole bundle stains under which the entire skeletal muscle tissue will be imaged. Another procedure would be to use whole bundle RNA isolation which allows for the analysis of RNA expression at some week to better understand the genetic effects the presence of supporting cell types may have on muscle development. Additionally, we may use qPCR to quantify the relative amounts of certain genes and which ones are expressed more than others.
Although it sounds simple, there are inherent limitations to such research. For one, cells are fickle and it is difficult to understand why an experiment may go awry or why cells may behave differently, and these fluctuations can only be mitigated through strict sterilization practices. Additionally, the growth cycles for engineered tissue take weeks to months to produce tissue, which is currently ineffective for clinical application. However, I am optimistic that the research I am doing will allow for us to take one step closer to the goal of quick tissue regeneration and integration to save victims suffering from dire wounds.