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. 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. Differential expression of the sodium channel auxiliary unit SCN3B was found and validated through patch-clamp measurements of sodium currents showing increased peak amplitude and window current in female hiPSC-CM. 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.