UTILIZZO DI CELLULE STAMINALI EMBRIONALI MURINE PER LA CREAZIONE DI UN PACEMAKER BIOLOGICO.

Mouse embryonic stem cells (ESCs) can be differentiated into spontaneously beating cell aggregates called Embryoid Bodies (EBs) which contain cells with functional and electrical properties of sinoatrial myocytes. These cells represent a small fraction of the subset of cardiomyocytes and an even a smaller fraction of the total cells composing the EBs. This work is aimed to isolate a homogeneous population of cardiac progenitors which specifically differentiate into pacemaker cardiomyocytes. We used transient expression, during cardiomyocyte differentiation, of CD166(or ALCAM, activated leukocyte cell adhesion molecule) to isolate cardiomyocyte progenitors from differentiating EBs. ESC have been cultured and differentiated as previously described1. CD166+ cells were sorted by flow cytometry and analyzed by quantitative RT-PCR, immunofluorescence and electrophysiology analyses. The data show that CD166+ cells isolated from 8 days-old EBs express significantly higher levels of cardiac genes (sarcomeric α-actinin, Mef2c, cTnI, Gata4) than both CD166- and undifferentiated ES cells. Our data also show that CD166+ cells express transcription factors involved in the generation of the conduction system (Shox2, Tbx18, Tbx3 and Isl-1) and genes typical of pacemaker myocytes (HCN4, HCN1, ssTnI) while expressing very low levels of typical ventricular genes (Nkx2-5, Cx43, HCN2). When CD166+ cells are allowed to re-aggregate for 24h in suspension and are then plated, they form a layer of synchronously beating cells that generate spontaneous action potentials modulated by autonomic neurotransmitters. Importantly, these cells can be maintained in culture for more than four weeks without losing their pacemaker activity. In conclusion our data show that ES-derived CD166+ cells isolated from differentiating EBs represent a population of cardiomyocytes, with the molecular and functional features of pacemaker cells, that can be cultured, in vitro, for long periods and can thus represent a suitable cellular pacemaker in cell therapy interventions.