| Abstract |
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.
|
