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

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a valuable cell type for studying human cardiac health and disease in vitro. However, it is not known whether hiPSC-CMs 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-CMs 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 3 female and 3 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-CMs and maintained them in basal media without exogenous hormones. Functional assessment of CMs showed enhanced calcium handling in female-derived hiPSC-CMs relative to male. Bulk RNA sequencing revealed over 300 differentially expressed genes (DEGs) between male and female hiPSC-CMs. Gene ontology analysis of DEGs 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-CMs. 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 CM function.

Keywords: adhesion receptors; calcium flux; cardiac; iPS; induced pluripotent stem cells.

Graphical Abstract

Figure 1.

Female human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have enhanced calcium-handling properties. (A) Schematic depicting the hiPSC-CM differentiation, purification, and the timepoints of CM purity assessment, RNA collection, patch-clamp, and calcium-handling data acquisition. (B) Representative flow cytometry data showing the cells on forward scatter (FSC) vs CM marker, cardiac troponin T (cTnT) on the x-axis. (C) Quantification of the percent CMs (cTnT+) by line and (D) by sex where the bar represents the average between n = 3 independent experiments across all 6 lines. Each dot represents the given cTnT+ (%) for one replicate. (E) Representative paced calcium handling trace (from M4L1-CM) showing some of the parameters assessed, such as the time to peak (t_p), the maximum amplitude (Max), and the maximum upstroke (V_up) and downstroke (V_down) velocities. Calcium transient (CaT) of the male and female hiPSC-CM bulk monolayers showing the (F) inter-spike interval (ISI), (G) time to peak, (H) maximum amplitude, and the CaT (I) upstroke and (J) downstroke. Box and whisker plots of the spontaneous CaT parameters, the (K) ISI, (L) the coefficient of variance (CV) of the ISI, showing the regularity of hiPSC-CM spontaneous CaT, and the (M) time to peak, for the male and female hiPSC-CMs. For (F-M), the box and whisker plots represent the median, upper, and lower quartiles while the (+) represents the mean. (F-M) The data are the average of 9 videos across 3 technical replicates for 3 independent experiments from all 3 female and all 3 male hiPSCs. Statistical comparisons were made using a pairwise Student’s t-test with *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Figure 2.

Bulk RNA sequencing reveals genetic differences between female and male human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). (A) Heat map of hiPSC-CM gene expression from 3 independent experiments for all 6 hiPSC lines. A dendrogram at the top of the heatmap shows separate hierarchical clustering of male and female hiPSC-CMs based on gene expression patterns. (B) Principal component analysis (PCA) plot showing separate clustering of male and female hiPSC-CMs, where each dot is one independent RNA sequencing replicate. (C) Volcano plot representing differentially expressed genes (DEGs) between male and female hiPSC-CMs. (D) Volcano plot analogous to (C) with non-Y-linked genes highlighted on the volcano plot. (E) Dot plot showing enriched gene ontology (GO) biological process (BP) pathways from an overrepresentation analysis of the DEGs between the male and female hiPSC-CMs. The dot size indicates the number of genes linked to each enriched pathway, while the dot color reflects the statistical significance of the enrichment. (F) Hallmark gene set enrichment dot plot highlighting gene sets that are suppressed in male hiPSC-CMs compared to the female hiPSC-CMs. The dot size signifies the number of genes associated with enrichment, and the dot color denotes the level of statistical significance.

Figure 3.

Differential ion channel-associated gene expression in male and female human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Graphs showing the expression level in counts per million (CPM) for the ion channel genes (A) sodium voltage-gated channel 3 B (SCN3B), (B) potassium inwardly rectifying channel subfamily J member 12 (KCNJ12), (C) potassium voltage-gated channel subfamily A member 7 (KCNA7), and (D) potassium voltage-gated channel subfamily E regulatory subunit (KCNE1). Sodium current recordings from hiPSC-CM patch-clamp are presented as (E) sodium current traces from male and female hiPSC-CMs, (F) current-voltage (I-V) curves, and (G) normalized activation and inactivation curves. (H) Immunocytochemistry for SCN3B (red), cardiac troponin T (cTnT: green) to mark cardiomyocytes, and DAPI (blue) to mark nuclei. Scale 50 μm. (I) Quantification of SCN3B area normalized to the DAPI count. For (A-D), the bar represents the average ± SD and each dot is the normalized expression level in CPM for one independent experiment with n = 3 replicates per lines and 3 hiPSC-CM lines per sex. Statistical significance designations are based on an Edge Test of raw reads and the false discovery rate corrected P-value. The test used to determine differentially expressed genes (DEGs). Where *P < .05, **P < .01, ***P < .001, and ****P < .0001. For (F-G), the data are presented as mean ± SEM. n = 10 for each group across 2 batches from 2 female and 2 male lines. A 2-sample Student’s t-test was used for statistical analysis with *P < .05. For (I), each data point represents the average of 5 fields of view across one well, and 2 wells were imaged per biologic replicate for all 6 lines. A 2-sample t-test was used for statistical analysis. For all bar graphs, ○: F5L1 and M4L1; ■: F6L1 and M7L1; and ▲: F7L1 and M9L1.