Variability and sexual dimorphism in human induced pluripotent stem cell derived cardiomyocytes.

Abstract

32 of every 100 global deaths are caused by cardiovascular diseases (CVD) [World Health Organization, CVD Fact Sheet, 2021]. Most mammalian research has been in male cell lines and subjects for over a hundred years which lead to sex related health inequities which are particularly acute for CVD since women have different symptoms than men, and respond differently to CVD treatment [Pierre, Frontiers in Physiology, 2022]. Only in the last ∼10 years have female subjects been required in experimental research studies [Miller, FASEB, 2016]. Human induced Pluripotent Stem Cell derived Cardiomyocytes (hiPSC-CMs) are an emerging model for the human heart but their sex differences remain understudied. For example, incomplete X chromosome inactivation (XCI) causes many differentially expressed genes between male and female cell lines even in the absence of sex hormones. We characterized the variability and sexual dimorphism of functional phenotypes in hiPSC-CMs using Traction Force Microscopy (TFM) to quantify contractile force generation. Some of this data was adapted from previously published work where we used standard differentiation and culture protocols for 3 female and 4 male cell lines and analyzed >40 cells for each line [Chirikian, JMCC, 2022]. We cultured hiPSC-CM on physiologically-relevant 10-kPa hydrogel substrates patterned with 1500 µm2 rectangular “islands” of Matrigel to constrain cell shapes to a 7:1 aspect ratio. Through regression analysis we observed statistically significant differences in contractile dynamics: male cell force was +19.5% (11%-28.5%), p = 0.0139, and spread area: Af = 823 +/- 17.32 (µm2) vs. Am = 756 +/- 21.64 (µm2), p = 0.0018. In ongoing studies, we are increasing the number of cells lines for each sex and evaluating the effect of estrogen and testosterone added to maintenance media.