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Human‐induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes have many promising applications, including the regeneration of injured heart muscles, cardiovascular disease modeling, and drug cardiotoxicity screening. Current differentiation protocols yield a heterogeneous cell population that includes pluripotent stem cells and different cardiac subtypes (pacemaking and contractile cells). The ability to purify these cells and obtain well‐defined, controlled cell compositions is important for many downstream applications; however, there is currently no established and reliable method to identify hiPSC‐derived cardiomyocytes and their subtypes. Here, we demonstrate that second harmonic generation (SHG) signals generated directly from the myosin rod bundles can be a label‐free, intrinsic optical marker for identifying hiPSC‐derived cardiomyocytes. A direct correlation between SHG signal intensity and cardiac subtype is observed, with pacemaker‐like cells typically exhibiting ~70% less signal strength than atrial‐ and ventricular‐like cardiomyocytes. These findings suggest that pacemaker‐like cells can be separated from the heterogeneous population by choosing an SHG intensity threshold criteria. This work lays the foundation for developing an SHG‐based high‐throughput flow sorter for purifying hiPSC‐derived cardiomyocytes and their subtypes.

An intrinsic, label‐free signal for identifying stem cell‐derived cardiomyocyte subtype