In Vitro Recapitulation of Cardiac Developmental Microenvironment for Biomimetic Maturation of Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Cardiovascular diseases are a leading cause of death worldwide which mostly result in cardiomyocytes (CM) loss. CM death during myocardial infarction and heart failure causes clinical challenges due to the heart inability to self-re-pair. Hence, the advent of human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) which can truly differentiate into CM is a promising tool for clinical and research applications. hPSC-CM, however, show neonatal-like phenotype which structurally and functionally differs from adult CM. Several maturation strategies have been applied to encourage the adult-like phenotype in CM and it is thought that recapitulating the physiological microenvironment for native CM allows them to fully mature. In this project, we combined three maturation ap-proaches, including the use of a three-dimensional (3D) plat-form, time in culture and co-culturing with endothelial cells. We aimed to investigate if co-culture and time in culture influence human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) calcium (Ca2+) cycling and metabolic function to better resemble adult-like CM.Materials and Methods: We co-cultured early (day 17-20) and late (day 45-52) hiPSC-CM with human umbilical vein en-dothelial cells (HUVEC) in 3D fibrin hydrogels for 7 days. Im-munostaining, mitochondrial membrane potential assay, Sea-horse assay and optical mapping were performed to investigate whether co-culture with HUVEC and/or time in culture altered hiPSC-CM functional properties.Results: We observed that the hiPSC-CM spontaneous beat-ing rate was significantly reduced in co-cultures regardless of age. Furthermore, HUVEC significantly decreased hiPSC-CM mitochondrial membrane potential while time in culture had the opposite effect. There was not significant difference in the oxy-gen consumption rate among different conditions. Finally, our data showed that HUVEC prolonged hiPSC-CM Ca2+ transients in late co-cultures.Conclusion: In conclusion, our data indicate that the combi-nation of the above-mentioned maturation strategies manipu-late hiPSC-CM mitochondrial and Ca2+ handling properties; however, this does not enable them to completely resemble the adult- like phenotype and other factors need to be also in-cluded to render these cells more suitable for heart regenera-tion strategies.

Keywords:

Human Induced Pluripotent Stem Cell , Maturation , Three-Dimensional Culture , Time in Culture , Co-Culture