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

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.

Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques

“…Our results suggest that, across three hiPSC lines, low (3 μM) Chiron concentrations with higher seeding density groups performed better than lower seeding density at any higher Chiron concentration. This is in agreement with findings from Laco et al that 3 μM Chiron in low S/G2/M populations (high density) produced roughly 40% pure hESC-cardiomyocyte populations while in high S/G2/M populations (low density), produced <5% pure hESC-cardiomyocyte populations [11]. Interestingly, the study reports the biggest difference in differentiation outcomes based on both Chiron concentration and S/G2/M levels between 5 μM and 6 μM Chiron.…”

“…concentration of Chiron in hPSC-cardiomyocyte differentiation depends on the proportion of the hPSC population in the S/G2/M phase of the cell cycle [11]. Additionally, the group showed that the confluency of hPSCs in culture correlates with the distribution of cell cycle stages in the population.…”

“…Cardiac differentiation is initiated with the application of a GSK3 inhibitor to activate Wnt signaling [7], which has been previously optimized at a concentration of 6 μM when using CHIR99027 (Chiron) by different groups [3,4,[8][9][10]. The concentration of GSK3 inhibitor required to initiate the mesodermal lineage depends most significantly on the proportion of induced pluripotent or embryonic stem cells (here on referred to as "hPSCs") in the S/G2/M stage of the cell cycle [11]. This proportion, in turn, depends primarily on cell density, colony size, and time in culture [12].…”

Computational fluid dynamics modeling, a novel, and effective approach for developing scalable cell therapy manufacturing processes

“…Based on these results, for a complex differentiation process, there are additional factors that need to be considered during the differentiation other than solely the hydrodynamic environment. Laco et al () recently published on the inconsistencies and low reproducibility observed with cardiac differentiation. It was observed that the cell cycle profile could influence the effects of GSK3β inhibition (i.e., CIHR99021) on cardiac differentiation and cell death.…”

Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity

“…It's well known that PSCs proliferative log phase is required for the best results in many differentiation protocols such as cardiac ones. [36] So, tracking cell doubling rates during their maintenance to avoid predictable high confluence and so cell cycle stuck is strongly desirable and it can be easily performable by PDT calculation and cell confluence monitoring as routine of culturing. Thus, we have assessed population doubling time (PDT) and cell confluence from passage 20 to 60, a time range that corresponded to approximately 120 days of cultivation.9…”