Abstract
Human induced pluripotent stem cell derived cardiomyocytes (hiCMs) exhibit a fetal phenotype, but tools to quantify their relative immaturity are scarce. We reasoned that, during myocyte specification, cells progress through myofibrillogenesis as force-generating units, known as sarcomeres, self-assemble along the cell cytoskeleton. Therefore, we developed image processing techniques to quantitatively score myocyte structural phenotypes by the increasing degree of organization and alignment that sarcomeres acquire during myofibrillogenesis. Since this is a highly conserved process, quantifications obtained from α-actinin immunostains in rodent and hiCMs can be compared. Utilizing these metrics we quantitatively showed that hiCMs patterned on square fibronectin islands had significantly under-developed contractile architecture, in agreement with the qualitative observation that these cells retain a more migratory cytoskeleton. Furthermore, we trained thee independent machine learning algorithms on over 100 α-actin immunostains from engineered primary cardiac tissues at 6, 24 and 48 hours after seeding. These preparations were taken to represent differentiated, immature and mature structural architectures, respectively. After training,[[Unable to Display Character: ]]α-actinin immunostains of hiPS-derived cardiac tissues were unbiasedly analyzed by these classifiers. The results indicated that ~30% of cells exhibited cytoskeletal architectures similar to those of mature myocytes and that treatment with commercially available small molecules influence hiCM structural maturation. In conclusion, we provided metrics to assess the organization of the contractile cytoskeleton in primary and stem cell-derived cardiomyocytes and to unbiasedly quantify their maturation.