Unraveling the Inconsistencies of Cardiac Differentiation Efficiency Induced by the GSK3β Inhibitor CHIR99021 in Human Pluripotent Stem Cells

Cardiac differentiation efficiency is hampered by inconsistencies and low reproducibility. We analyzed the differentiation process of multiple human pluripotent stem cell (hPSC) lines in response to dynamic GSK3β inhibition under varying cell culture conditions. hPSCs showed strong differences in cell-cycle profiles with varying culture confluency. hPSCs with a higher percentage of cells in the G1 phase of the cell cycle exhibited cell death and required lower doses of GSK3β inhibitors to induce cardiac differentiation. GSK3β inhibition initiated cell-cycle progression via cyclin D1 and modulated both Wnt signaling and the transcription factor (TCF) levels, resulting in accelerated or delayed mesoderm differentiation. The TCF levels were key regulators during hPSC differentiation with CHIR99021. Our results explain how differences in hPSC lines and culture conditions impact cell death and cardiac differentiation. By analyzing the cell cycle, we were able to select for highly cardiogenic hPSC lines and increase the experimental reproducibility by predicting differentiation outcomes. •Lineage variety and cell culture density affect the cell cycle in hPSCs•CHIR99021 is cytotoxic to hPSCs with reduced S/G2/M cell-cycle phases•Cardiac differentiation reproducibility depends on cell-cycle consistency in hPSCs•Cell cycle and TCF protein levels modulate CHIR99021-induced differentiation In this article, Laco and colleagues show that human pluripotent stem cells are characterized by individual cell-cycle profiles. Changes in cell-cycle profiles, mainly due to cell culture density, affect Wnt pathway-induced cardiac differentiation and cell death when GSK3β inhibitors are applied. We show that cell-cycle validation is crucial for stem cell quality and experimental reproducibility.