JAK2 as a surface marker for enrichment of human pluripotent stem cells-derived ventricular cardiomyocytes
The generation and purification of cardiomyocyte sub-population are critical for precise modelling of cardiac diseases, drug screening, and regenerative medicine due to their distinct phenotypic and functional characteristics. Therefore, identifying surface markers for cardiomyocyte subtypes that facilitate live cell sorting of homogenous population of cardiomyocytes is of utmost importance for successful translation of cardiovascular research and therapeutic applications. Using MARIS method coupled with RNA sequencing analysis on MLC2A- and MLC2V-expressing cardiomyocytes, we identified JAK2 as a potential cell surface marker for purifying ventricular cardiomyocytes. We demonstrated that that SIRPA+/JAK2+ population sorted expressed high levels of ventricular-specific marker and had electrophysiological properties that corresponded to ventricular-like cardiomyocytes. Functionally, SIRPA+/JAK2+ cardiomyocytes do not response to vernakalant, an atrial-selective anti-arrhythmic agent, confirming their identity as ventricular-like cells. Overall design: We used MARIS to isolate SIRPA+ CMs based on intracellular MLC2A or MLC2V expression and identified potential surface markers for ventricular specification through RNA sequencing analysis. To validate the effectiveness of these surface markers, we performed immunostaining and western blot analysis to measure MLC2A and MLC2V protein expressions in SIRPA+ cardiomyocytes that were either positive or negative for the putative surface markers. We then characterized the electrophysiological properties of surface marker-sorted cardiomyocytes by measuring cellular calcium transient using fluo-4 AM. For functional validation, we investigated the response of the surface marker-sorted cardiomyocytes to vernakalant, an atrial-selective anti-arrhythmic agent.