Optimization of Small-Molecule Wnt Pathway Activation with CHIR99021 for Human Induced Pluripotent Stem Cell...

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising tool to model and investigate human heart disease in vitro. To achieve this goal, differentiation protocols must be optimized to produce pure populations of somatic cells, such as cardiomyocytes (CMs). CHIR99021 (CHIR) is a small molecule that inhibits the enzyme glycogen synthase kinase-3 beta (GSK-3β), activating the Wnt/β-catenin pathway and genes involved in cardiac cell growth and differentiation. Therefore, CHIR is commonly used to initiate directed CM differentiation from hiPSCs. However, the optimal concentration of CHIR varies for different cell lines, making maximum differentiation efficiency a significant hurdle. Mutations in Rotatin (RTTN), a gene encoding a centrosomal protein, have been linked to defective heart maturation and cardiovascular disease, but this protein and other centrosomal proteins must be explored further using hiPSC-CMs. Based on conventional lab protocol, we hypothesized that 12 μM CHIR would have the highest differentiation efficiency. In this study, we used immunostaining protocols to stain and image CC2 CMs (wild-type cells) and LVIP CMs (containing a patient-specific compound heterozygous RTTN mutation), allowing protein localization to be observed through imaging and manual counting. This study suggests a concentration of 6 μM CHIR promotes optimal CM differentiation efficiency for both CC2 and LVIP hiPSCs, challenging our hypothesis. The findings of this study are crucial to enhance understanding regarding the optimal differentiation of cardiomyocytes from hiPSCs. Moreover, they constitute a step toward future research into the role of centrosomal proteins in cardiovascular disease.