Developing a Tool to Confirm the Successful Differentiation of Human Pluripotent Stem Cells into Cardiomyocytes
Heart disease has been the leading cause of death in the United States since 1921 (Greenlund et al., 2006). However, age-adjusted cardiovascular disease death rates have declined by 55% since 1950, a trend that is in part due to improvements in treatment (Greenlund et al., 2006). Researchers interested in developing effective heart disease treatments have begun using human myocardial cells derived from induced pluripotent stem cells (iPSCs) to build in vitro heart disease models, screen for new cardiovascular drugs or drug side effects, and study individualized cardiac therapy (Sinnecker et al., 2012). The future clinical use of iPSCs in cell therapy for cardiovascular disorders is also incredibly promising (Sinnecker et al., 2012). However, when differentiating iPSCs into cardiomyocytes, researchers currently have no tools available to confirm whether the differentiation was successful. The goal of this project was to create and test a tool that can confirm and quantify the successful differentiation of iPSCs into cardiomyocytes by coupling the GCaMP8 calcium indicator and the cardiomyocyte-specific promoter alpha myosin heavy chain (αMHC). A release of calcium ions triggers cardiomyocyte contraction (Marks, 2003). When calcium ions bind to GCaMP molecules a conformational change occurs that results in an increase in green fluorescence (Shui et al., 2014). Therefore, an αMHC-GCaMP8 cardiomyocyte cell line should produce green fluorescence during contraction. This project demonstrated the use of Lipofectamine 3000 to transfect human iPSCs with an αMHC-GCaMP8-iCAG insertion plasmid and the successful differentiation of transfected iPSCs into cardiomyocytes, and it suggests that differentiated cardiomyocytes fluoresce.
Biological sciences honors program; cardiomyocytes; induced pluripotent stem cells; differentiation; transgenics; fluorescence
B.A., Biological Sciences
Bachelor of Arts
dissertation or thesis