Abstract
SummaryCardiac 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.
Highlights
Glycogen synthase kinase-3b (GSK3b) has multiple cellular substrates, and they play strategic roles in various essential physiological processes, such as development, the cell cycle, and apoptosis
We studied the effect of CHIR induction in human pluripotent stem cell (hPSC) lines to understand its dynamics and facilitate mesoderm formation resulting in cardiac differentiation
We observed in repeated HES3 differentiation experiments (n = 22, >160 embryoid body (EB)) using initial cell culture conditions, such as 60%–80% cell confluency, 85% NANOG and OCT4a cell population expression by flow cytometry, and a constant number of seeding cells (15,000 cells/EB), that a wide range of initial EB sizes were formed (0.025–0.225 mm2 or 2,500–22,500 cells/EB) on day 1
Summary
Glycogen synthase kinase-3b (GSK3b) has multiple cellular substrates, and they play strategic roles in various essential physiological processes, such as development, the cell cycle, and apoptosis. Nuclear b-catenin forms a complex with transcription factor (TCF) proteins to activate the Wnt pathway gene targets (McCubrey et al, 2014) These Wnt gene targets affect the expression of pluripotency and developmental factors associated with the primitive streak and the germ layers (Hodar et al, 2010). The reproducibility of the protocol requires cell line- and cell culture-dependent optimization and can lead to heterogeneous differentiation results (Sepac et al, 2012). It is not clear how a single transient induction with a GSK3b inhibitor is able to direct highly efficient lineage specification toward cardiomyocytes. We studied the effect of CHIR induction in hPSC lines to understand its dynamics and facilitate mesoderm formation resulting in cardiac differentiation
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