Sex dimorphism is present in human stem cell-derived cardiomyocytes without the addition of exogenous hormones

Introduction: Cardiovascular disease (CVD) is the leading cause of mortality and morbidity globally. When developing models of CVD, consideration of sex dimorphism is essential as clinical and animal studies have revealed stark sex differences in CVD onset and progression. Sex dimorphism in CVD is expansive and encompasses ischemic heart disease, responses to ischemia-reperfusion injury, cardiac remodeling in response to pressure overload, and the occurrence of both innate and drug-acquired arrhythmias. Sex differences have also been reported in heart development, as well as heart function in healthy individuals. These have been reported both at the organ and cellular level in human and animal studies.
Over the past decade stem cells, particularly human induced pluripotent stem cells (hiPSC), have become a source for creating in vitro model systems to study human CVD and health. 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.

Materials and

Methods: Here we generated three female and three male hiPSC-lines from adult left ventricular cardiac fibroblasts (LVCFs) as a resource for studying sex differences in in vitro cardiac models. The LVCFs were reprogrammed using CytoTune 2.0 Sendai Virus (SeV) methodology from Thermo Fisher Scientific. The lines were assessed for pluripotency markers and SeV depletion using q-RT-PCR. Pluripotency was additionally assessed at the protein level via flow cytometry and immunocytochemistry (ICC). All six lines were karyotyped using G-band karyotyping determining a normal XX or XY karyotype in all lines.

All six hiPSC-lines were then differentiated into hiPSC-CM using small molecule WNT modulation and purified using a metabolic selection medium. The CM purity was assessed using flow cytometry for cardiac troponin T (cTnT). At day 40 RNA lysis was obtained and at day 60 the calcium handling ability of the hiPSC-CM was assessed using the calcium-sensitive dye Rhod-2AM. The calcium transient data was acquired under both paced (1 Hz), and spontaneous conditions. An in-house MATLAB code was used to determine the calcium transient time to peak, maximum amplitude, and the upstroke and downstroke velocities. In the spontaneous condition beating irregularity was assessed via the coefficient of variation (CV) of the inter-spike-interval (ISI). For RNA sequencing the bulk RNA lysates were purified and high RNA quality control was done to ensure RNA integrity. Libraries were created using Illumina’s TruSeq RNA Sample Preparation Kit and then sequenced, normalized, and analyzed using the Absolute Fold Change and FDR corrected p < 0.05 of normalized raw reads.

Results, Conclusions, and Discussions: The hiPSC-lines from the female and male LVCFs were determined to be highly pluripotent via q-RT-PCR, ICC, and flow cytometry (Figure 1). The female and male hiPSC-CM differentiated and purified into a highly pure CM population (Figure 2A-D). Functional assessment of CM showed enhanced calcium handling in female-derived hiPSC-CM relative to male as seen through a net decrease in the time to peak and an increase in the maximum amplitude and upstroke velocity (Figure 2E-J). The female hiPSC-CM had a slightly higher arrhythmic tendency as seen through an increase in the CV of the ISI (Figure 2K-M).

The female and male hiPSC-CM were genetically distinct from each other as seen through separate hierarchical and PCA clustering of the RNA sequencing data (Figure 3A-B) Bulk RNA sequencing revealed over 300 differentially expressed genes (DEG) between male and female hiPSC-CM (Figure 3C-D). Some of the DEG are X and Y-linked genes and many are implicated in cardiac health and disease including sodium and potassium channels which could account for net differences in calcium handling shown here (Figure 3E-H). 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 (Figure 3I-J).

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. Here we show that hiPSC-CM exhibit sex dimorphism even without the addition of exogenous hormones. This encompasses differences in calcium handling capabilities and differences in the gene expression of critical cardiac ion channels. These results suggest that sex should be included as a variable in benchtop research on CVD and cardiac health when utilizing hiPSC-CM.

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