Optimized conditions for the supplementation of human-induced pluripotent stem cell cultures with a GSK-3 inhibitor during embryoid body formation with the aim of inducing differentiation into mesodermal and cardiac lineage.

We optimized the conditions for the differentiation of human induced pluripotent stem cells (hiPSCs) into mesoderm lineage-committed cells by supplementing the cultures with CHIR, a selective GSK-3 inhibitor, during embryoid body (EB) formation. In vitro treatment with 4 µM CHIR during the late 2 days of a 4-day suspension culture period was most effective at promoting mesodermal differentiation. The resulting EBs showed a significant increase in the expression levels of mesoderm-associated genes (WNT3A, T, DKK1, GATA4, FOXC1, and MESP1) and a maintenance of OCT3/4 and NANOG expressions. Upon subsequent differentiation into a cardiac cell lineage, these EBs were shown to generate contractile cardiomyocytes. When shortening the CHIR treatment period to 1 day, the resulting EBs showed reduced expression of mesoderm-associated genes in comparison to the 2-day CHIR treatment. In particular, the expression level of FOXC1 in the 1-day CHIR-treated EBs was much lower than that of the 2-day CHIR-treated EBs. When the treatment period with CHIR was extended to 4 days, the resulting EBs presented significantly reduced expression of WNT3A, OCT3/4, and NANOG upon CHIR concentrations above 4 µM. Similarly, when CHIR treatment was conducted after the formation of EBs, the effectiveness of the GSK-3 inhibitor was reduced compared to a treatment performed during EB formation. Our results indicate that spatiotemporal constraints associated with EB formation, i.e., three-dimensional structuration and cell development in EBs, should be taken into account when designing EB formation-based differentiation protocol involving CHIR treatment.

Optimization of the treatment conditions with glycogen synthase kinase-3 inhibitor towards enhancing the proliferation of human induced pluripotent stem cells while maintaining an undifferentiated state under feeder-free conditions.

The small molecule inhibitor CHIR99021 (CHIR) is well known as a selective glycogen synthase kinase-3 inhibitor. The purpose of our study was to optimize the conditions of CHIR supplementation that will enhance the proliferation of human induced pluripotent stem cells (hiPSCs) while maintaining their undifferentiated state under feeder-free conditions in adherent cultures for 4 days. Our results revealed that both of the timing and concentration of CHIR affected cell behaviors of hiPSCs, such as colony formation, cell proliferation, and differentiation. The addition of 1-3 µM CHIR to hiPSCs cultures in the late 2-day period of a 4-day cultivation was effective in enhancing cell proliferation. Treatment with 3 µM CHIR significantly enhanced cell proliferation, but led to differentiation of hiPSCs when the treatment was carried out over 4 days. Treatment with higher concentration of CHIR was also conducive to deviating hiPSCs from their undifferentiated state. Treatment with 10 µM CHIR led to decreased expression of pluripotency-associated genes and increased level of mesoderm marker genes, but failed to provided any growth-promoting effect. Interestingly, when treatment with 1 µM CHIR was confined to the late 2-day period of a 4-day cultivation, cell proliferation was enhanced without detectable deviation from the undifferentiated state as evidenced by the expression levels of pluripotency-associated genes, e.g., OCT3/4, NANOG, SOX2, and REX1. Repeated use of 1 µM CHIR in subcultures provided no adverse effect on the proliferation of hiPSCs. Our results indicated that carefully designed CHIR treatment allows for enhanced proliferation of hiPSCs.