Healthy human induced pluripotent stem cell-derived cardiomyocytes exhibit sex dimorphism even without the addition of hormones

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a valuable cell type for studying human cardiac health and disease in vitro. However, it is not known whether hiPSC-CM display sex dimorphism and therefore whether sex should be incorporated as a biological variable in in vitro studies that include this cell type. To date, the vast majority of studies that utilize hiPSC-CM do not include both male and female sex nor stratify results based on sex because it is challenging to amass such a cohort of cells. Here we generated three female and three male hiPSC-lines from adult left ventricular cardiac fibroblasts as a resource for studying sex differences in in vitro cardiac models. We used this resource to generate hiPSC-CM and maintained them in basal media without exogenous hormones. Functional assessment of CM showed enhanced calcium handling in female-derived hiPSC-CM relative to male. Bulk RNA sequencing revealed over 300 differentially expressed genes (DEG) between male and female hiPSC-CM. Some of the DEG are X and Y-linked genes and many are implicated in cardiac health and disease including potassium channels which could account for net differences in calcium handling shown here. Gene ontology analysis of DEG showed distinct differences in pathways related to cardiac pathology including cell-cell adhesion, metabolic processes, and response to ischemic stress. These findings highlight the importance of considering sex as a variable when conducting studies to evaluate aspects of human cardiac health and disease related to cardiomyocyte function.