Mouse embryonic stem cells (mESCs) are known to differentite into all cardiac phenotypes, and may thus represent an important potential source of cells for cardiac regenerative therapies. Previous data, obtained in our laboratory, indicate that mESCs, differentiated through the formation of embryoid bodies (EBs) generate cells with functional and molecular characteriscics typical of cardiac pacemaker myocytes.

The isolation of these cells could provide a substrate suitable for the generation of ESC-based biological pacemakers. Since the HCN4 isoform is a specific marker of the cardiac conduction tissue, to facilitate the localization and isolation of a pacemaker myocytes, we have generated a mESC line stably expressing GFP under the transciptional control of a promoter region of the mouse HCN4 gene. The integration of the construct into the genome was confirmed by PCR analysis and the EBs generated with engeenered mESCs showed EGFP positive (EGFP+) cells in the beating area. Immunofluorescence experiments carried out in whole EBs and isolated cells confirmed the presence of the HCN4 protein, together with the cardiac markers in EGFP+ cells. Furthermore, preliminary electrophysiological data show that isolated EGFP+ cells present spontaneous action potentials and the I_f current. In conclusion our data show that the HCN4 promoter is able to drive GFP expression in differentiating mESC and EGFP+ myocytes present several properties typical of pacemaker myocytes.