Abstract 10982: Cardiac Cell Monolayer Derived from Human Pluripotent Stem Cells
Introduction: Stem cell research, including cardiac differentiation from human pluripotent stem cells, is fast-evolving field, especially important for the regenerative medicine. In order to advance research to the medical applications, it is crucially important to have as full knowledge of the differentiated tissue as possible. In our work, we study monolayers of stem cell-derived cardiac tissue as a model system for prediction of arrhythmogenesis.
Methods: Cardiomyocytes were differentiated from human induced pluripotent stem cells (253G1) for 3-4 weeks, purified and platted on tissue culture dishes (12 mm-diameter). They were confirmed as electrical syncytium using optical mapping with Ca2+ sensitive dye (Fluo-4, 50fps, at room temperature). Quantitative real time polymerase chain reaction (PCR) and immunostaining were used to analyze these monolayers. We measured relative gene expression levels compared to GAPDH at 2, 5, 7, 8, 14, and 28 days after seeding.
Results: Excitation wave propagation was detected in the cell monolayer from 2 to 21 days after seeding. Propagation wave velocity was dependent on the stimulation cycle length and lidocaine concentration. The mean wave velocity was 40.3, 26.6, and 22.8 mm/s when the lidocaine concentration was 0, 0.1, and 0.2 mM, respectively (n=18, One-way ANOVA P<0.01). Reentrant (spiral) wave propagation was detected in the cell layers when lidocaine was added while applying high-frequency stimulation (n=9/23). The size of unexcitable spiral core increased depending on the concentration of E-4031 (n=1) and nifekalant (n=5). There was significant difference of mean α-actinin positive area between the monolayer in which spiral wave was detected and the monolayer in which it could not be detected or disappeared easily (n=4 for each, 64.6% vs 83.3%, t test P=0.01). With quantitative PCR, the expression of HERG-1b increased, but that of α-MHC and Cav1.2 decreased during the time course. The expressions of cardiac troponin T, β-MHC, and Cx43 were present consistently.
Conclusions: We succeeded to create powerful tool to evaluate the arrhythmogenic potential of cardiac tissue derived from human pluripotent stem cells. Cardiac purity was important to prevent spiral wave propagation in our model system.
- © 2011 by American Heart Association, Inc.