Abstract 12398: CD151 Expression Reflects Different Features in Cardiac Subtype Specification From Human Pluripotent Stem Cells

Abstract

Introduction: In conventional cardiac differentiation methods, a heterogeneous cardiac population is generated from human induced pluripotent stem cells (hiPSCs). Purifying ventricular- or atrial cardiomyocytes (VCMs or ACMs, respectably) is valuable for drug discovery for region-specific heart diseases. Previously, we have identified a novel marker, CD151, which can separate advanced specified subtypes; CD151 high VCMs in ventricular inducing condition (VIC) and CD151 low ACMs in atrial inducing condition (AIC). Although CD151 is known as a cell adhesion molecule, the function in cardiac subtype specification is unclear. In this study, we addressed the mechanisms of how CD151 distinguishes cardiomyocyte subtypes in cardiac differentiation from hiPSCs. Methods: We performed RNA sequencing in VIC- and AIC- CD151 high/low CMs. Pathway analysis using differential expression genes between CD151 high/low CMs was conducted to identify prominent pathways in subtype specification. Results: In VIC, mitosis-related genes were enriched in CD151 high VCMs. To clarify whether those genes affect cell proliferation or multi-nucleation, we performed the flow cytometric analysis of CD151 high/low VCMs with KI-67 proliferation marker and Hoechst staining. Although the percentage of KI67-positive cells was almost the same, binucleated cells were significantly more in CD151 high VCMs than in CD151 low VCMs. In AIC, transcriptome analysis suggested that Notch signaling was activated in CD151 high ACMs. To reveal that Notch signaling inhibits atrial specification, we analyzed ACMs treated with LY411575, a Notch signaling inhibitor. LY411575 induced atrial marker gene expression and increased the number of cells showing action potentials of ACMs. CD151 low ACMs after LY411575 treatment contained ACMs with the highest efficiency. Conclusion: Our results demonstrate that the CD151 expression indicates mitotic activity in VCMs. CD151 high VCMs are more advanced specified VCMs with binucleation without cell proliferation. On the other hand, in ACMs, CD151 works as an indicator of Notch signaling, which suppresses atrial specification. Our study provides a novel approach to generating and purifying hiPSCs-ACMs by Notch inhibition and CD151 separation.