Fig. 1

Differentiation of hiPSC colonies to pacemaker cell clusters. a Immunostaining of undifferentiated hiPSC shows typical morphology and provides evidence of pluripotency markers. b Positive immunostaining of undifferentiated hiPSC colonies for pluripotency markers (top) and absence of staining in clusters differentiated by co-culture (dhiPSC) (bottom). c Transcription of pluripotency markers in hRA, undifferentiated hiPSC, dhiPSC, and PCC. d Differentiation/maturation protocol: hiPSC and END-2 cells were co-cultured for 10–12 days (dhiPSC) followed by transfer to an FBS-enriched culture medium for further differentiation/maturation (PCC; altogether 56 days). Subsequently, functional analysis was undertaken in a co-culture model with neonatal rat ventricular myocytes (NRVM) (28 days). e Proliferation assay of undifferentiated hiPSC (top) and throughout the differentiation/maturation process (bottom). Scale bars = 100 μm. Data are provided as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, versus hRA; comparison between multiple groups was performed using one-way ANOVA followed by a Tukey post-hoc test. Statistical significance compared to hiPSC is not indicated (p < 0.01 in each panel). hRA human right atrium (dark green), hiPSC human induced pluripotent stem cells (red), dhiPSC co-culture differentiated hiPSC (blue), PCC pacemaker cell clusters (purple), RLU relative light units