Comparison of Non-Invasive Video-Based Tools in the Analysis of iPSC-CM Contractility

Induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes (CMs) offer tremendous opportunities for cardiac disease modeling and advancements in precision medicine, particularly in high-throughput drug screening and functional assays. This study focuses on optimizing noninvasive methods to measure iPSC-CM contractility, a critical indicator of cardiac function, in a simple two-dimensional video based setting using two open-source macros for ImageJ, MYOCYTER and MUSCLEMOTION, and evaluating against a third, Adaptive Reference-Digital Image Correlation (AR-DIC), MATLAB based approach. These tools analyze high-speed video data to assess contraction and relaxation parameters. MYOCYTER improves precision by allowing customizable parameters, minimizing disturbances and optimizing thresholding, while MUSCLEMOTION provides rapid and comparative assessments of motion. The AR-DIC method enables absolute displacement measurements of cell motion. The study leverages a disease model for Hypoplastic Left Heart Syndrome (HLHS), a severe complex congenital heart defect associated with MYH6 gene variants, which disrupt sarcomere structure and contractility in iPSC-CMs. Gene editing using CRISPR/Cas9 previously demonstrated phenotype rescue, establishing MYH6 iPSC-CMs as a robust HLHS model. To evaluate analytical tools, a team of high school students was trained to process longitudinal iPSC-CM videos (Day 20-30 post-differentiation) using MYOCYTER, and compared with analyses by MUSCLEMOTION, and AR-DIC. Cross-validation and 10% overlapping analyses will ensure data reliability. The study aims to evaluate iPSC-CM maturation, functional phenotypes under varying magnifications, examine the effects of pharmaceutical compounds, and compare the strengths and limitations of the tested tools. Findings will inform advancements in cardiac phenotyping and drug discovery, highlighting the role of robust analytical methods in precision medicine. Collaborative efforts of Brookfield East High School SMART Team and the Medical College of Wisconsin. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.