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Abstract Background Human cardiac stem cells expressing the W8B2 marker (W8B2+ CSCs) were recently identified and proposed as a new model of multipotent CSCs capable of differentiating into smooth muscle cells, endothelial cells and immature myocytes. Nevertheless, no characterization of ion channel or calcium activity during the differentiation of these stem cells has been reported. Methods The objectives of this study were thus to analyze (using the TaqMan Low-Density Array technique) the gene profile of W8B2+ CSCs pertaining to the regulation of ion channels, transporters and other players involved in the calcium homeostasis of these cells. We also analyzed spontaneous calcium activity (via the GCaMP calcium probe) during the in vitro differentiation of W8B2+ CSCs into cardiac myocytes. Results Our results show an entirely different electrophysiological genomic profile between W8B2+ CSCs before and after differentiation. Some specific nodal genes, such as Tbx3, HCN, ICaT, L, KV, and NCX, are overexpressed after this differentiation. In addition, we reveal spontaneous calcium activity or a calcium clock whose kinetics change during the differentiation process. A pharmacological study carried out on differentiated W8B2+ CSCs showed that the NCX exchanger and IP3 stores play a fundamental role in the generation of these calcium oscillations. Conclusions Taken together, the present results provide important information on ion channel expression and intrinsic calcium dynamics during the differentiation process of stem cells expressing the W8B2 marker.