CARATTERIZZAZIONE MOLECOLARE ATTRAVERSO ANALISI DI IMMUNOFLUORESCENZA DI CARDIOMIOCITI PACEMAKER DERIVATI DA CELLULE STAMINALI EMBRIONALI MURINE INGEGNERIZZATE

Introduction and Aim. Mouse embryonic stem cells (mESCs) are able to differentiate in vitro into many cell types derived from the three germ layers, including pacemaker cardiomyocytes. ESCs thus represent a potential cell source for generation of biological pacemakers repair. The applicability of ESCs in regenerative therapies requires the isolation of a pure population of differentiated cells in order to avoid the contamination with teratogenic undifferentiated ESCs. To identify ESC-derived pacemaker cardiomyocytes, we established a mESC line stably expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of the promoter of the HCN4 gene (pHCN4-EGFP). Although EGFP-positive spontaneously beating cells can be detected, non-beating EGFP-positive cells can be detected as well. The transmembrane protein CD166 is specifically expressed in the developing murine heart tube at embryonic day 8.5. Previous works have demonstrated that CD166 can be used as surface antigen to select a differentiated cell population enriched in cardiomyocytes. The aim of this work is to characterize the cardiac nature of CD166-positive and negative cell populations isolated from differentiating mESC cultures at day 7+1. Materials and Methods. pHCN4-EGFP mESCs-D3 were differentiated into embryoid bodies (EBs) by the “hanging drops” method. EBs at differentiation day 7+1 were dissociated into single cell-suspensions and sorted by flow cytometry. CD166+ and CD166 cells were plated in very low adhesion dish for 24 hours to facilitate their re-aggregation and then plated on cell culture dishes. Immunofluorescence experiments were carried out both on CD166+ and CD166 cell cultures. Samples were fixed in paraformaldehyde and labelled with antibodies against sarcomeric α-actinin to identify cardiomyocytes and against HCN4 and caveolin 3 to specifically detect cells with a pacemaker-like phenotype. Results. While CD166+ cells form spontaneously and synchronously beating aggregate, CD166 cells form EBs-like aggregates in which only very few cells show signs of automaticity. Immunofluorescence analysis of these aggregates demonstrate that 60,13 % of CD166+ cells expresses a contractile apparatus organized in sarcomeric-like structures, as shown by α-actinin staining. In contrast CD166 aggregates present a much lower degree of α-actinin staining (3,60 % of cells). To identify cardiac cells with pacemaker-like phenotype, double staining with anti-HCN4 and anti-α-actinin antibodies was carried out. In CD166+ cell population 42,77 % of α-actinin positive cell expresses HCN4 channels whereas in CD166 cell population the percentage is only 5,12 %. Moreover CD166+ cells also stained positive for caveolin 3, a structural protein of muscular caveolae, known to interact with pacemaker channels in both native SAN myocytes and ESC-derived pacemaker cells. Interestingly, most CD166+ cells were also EGFP-positive confirming a persistent activation of the HCN4 promoter in these cells and thus their pacemaker phenotype. Conclusions. Our data demonstrate that CD166 can be used, in a specific stage of differentiation, as a surface antigen to select a cell population enriched in cardiomyocytes presenting some peculiar features of sinoatrial pacemaker cells. For this reason, CD166+ cell population has the characteristics that made them a suitable cellular substrate for the creation of biological pacemakers.