Introduction:
Patient-specific human induced pluripotent stem cells (hiPSC)-derived cardiomyocytes (CMs) are increasingly used for
in vitro
disease modeling and drug screening, as well
in vivo
regenerative therapies. The cardiac differentiation efficacy of hiPSCs, together with the maturation level of generated CMs, are critical factors in achieving the required numbers of functional patient-specific cardiac muscle cells for clinical applications. Although extensive studies have improved the efficacy of differentiation and maturation processes, the role of cell sex in these processes has not been fully investigated.
Hypothesis:
Cell sex affects i) the cardiogenic differentiation efficacy of hiPSCs; and ii) maturation processes of hiPSC-CMs.
Methods and Results:
We have successfully and reproducibly fabricated patterned substrates recapitulating the 3D shape of mature CMs, using photolithography approaches, and demonstrated that the substrate could i) accelerate the differentiation of hiPSCs to CMs, and ii) facilitate maturation and functionality of immature hiPSC-CMs. Male and female hiPSCs, derived from human amniotic mesenchymal stem cells of male and female fetuses, were cultured onto flat (control)
vs.
patterned substrates. A total of 400 differentiation assays were conducted, 200 per each cell sex, on the flat (
n
= 100) and patterned (
n
= 100) substrates. A chemically defined approach was used to differentiate the cells toward CMs. On the flat (conventional) substrates, 59% of batches of male and 87% of batches of female hiPSCs differentiated into beating CMs (> 80%). On the patterned substrates, these numbers changed to 83% and 94% of successful differentiations for male and female hiPSCs, respectively. These results indicate the significant effect of substrate-mediated topographical cues on the cardiac differentiation yield of stem cells and the batch-to-batch variation. On both substrate types, female cells demonstrated significantly higher success rates of cardiac differentiation compared to the male cells. In addition, the CMs produced on the patterned substrates demonstrated higher purity than those created on the flat substrates both for male and female cells. Quantitative polymerase chain reaction (qPCR) was used to probe the male and female cell differences in expression of genes related to cardiac maturity, contractility, and Ca
2+
transport (TNNT2, MYH6, MYH7, and CACNA1c) and the outcomes revealed substantially greater expression levels of the maturation genes in differentiated female CMs cultured on the patterned substrates compared to the male cells.
Conclusions:
These results indicate that male and female hiPSCs and hiPSC-CMs respond differently to the identical substrates in terms of their differentiation and maturation efficacies.
Metabolic Regulation of Cardiac Differentiation and Maturation in Pluripotent Stem Cells: A Lesson from Heart Development
