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Insights into the expression of pacemaker‐specific markers in human induced pluripotent stem cell (hiPSC)‐derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC‐derived pacemaker‐like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker‐, atria‐, and ventricular‐like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch‐clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same‐single‐cell analysis for the first time revealed expression of proposed pacemaker‐specific markers—hyperpolarization‐activated cyclic nucleotide‐modulated (HCN)4 channel and Islet (Isl)1—at the protein level in all three hiPSC‐derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker‐like hiPSC‐derived cardiomyocytes than atrial‐ and ventricular‐like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker‐like hiPSC‐derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular‐like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC‐derived pacemaker‐like relative to ventricular‐like cardiomyocytes, these markers alone are insufficient in identifying hiPSC‐derived pacemaker‐like cardiomyocytes. S tem  C ells   2016;34:2670–2680

stem cells

Same‐Single‐Cell Analysis of Pacemaker‐Specific Markers in Human Induced Pluripotent Stem Cell‐Derived Cardiomyocyte Subtypes Classified by Electrophysiology