STUDIO DEL DIFFERENZIAMENTO CARDIACO DI CELLULE STAMINALI ISOLATE DA TESSUTI POST-NATALI

In recent years the stem cells are subject of intensive studies because they are able to differentiate in different types of cells and they can be use for regenerative therapies. In particular we are interested to the care of several acquired or genetic cardiac diseases that they are characterized by rhythm disturbances. These diseases include for example severe bradycardia, Sick Sinus Syndrome, atrio-ventricular block and heart block and often the pharmacological treatment is not always applicable. The our major object is to create a biological pacemaker, generally intended as cell substrates with the same features of native sinoatrial node (SAN) myocytes. So far, murine and human embryonic stem cells have been shown to differentiate into pacemaker myocytes, with electrical and molecular characteristics similar to those of SAN cells. Embryonic stem cells are at the moment one of the most promising cell sources for regenerative but they have different ethical and immunological problems. For this purpose we intend to study different type of adult stem cells and, in particular, a population of hematopoietic stem cells CD34+ isolated from umbilical cord blood and a class of vessel-associated clonogenic, self-renewing progenitor cells, called Mesoangioblasts, Mabs. In this work we investigated whether CD34+ endothelial progenitor cells acquire spontaneous beating and pacemaker properties by using a co-culture system onto neonatal cardiac. We found that the CD34+ cells acquire a spontaneously contractile phenotype but it were the result of fusion events between cardiac myocytes and CD34+-derived cells, as assessed by double color labeling experiment and high throughput FACS analysis. Furthermore the electrophysiological properties investigated did not show any difference between EGFP+ cells and neonatal cardiac myocytes, supporting the hypothesis that these autorhythmic cells derive indeed from a fusion events between electrically passive CD34+ cells with cardiac myocytes. Furthermore we have shown that ventricle-derived Mabs when cultured in low-serum medium spontaneously differentiate into cardiomyocytes that express typical cardiac ion channels. A distinct feature of cardiac MABs not described previously in other adult cardiac stem cells is the high spontaneous rate of cardiac differentiation, which allows the prospective use of these cells for systemic delivery and in vivo transplantation. Cardiac MABs express several factors involved in early cardiac differentiation, among these such as Isl-1, Tbx2, Tbx3, GATA-6 are especially relevant to the development of the cardiac conduction system. Upon differentiation, cardiac MABs often displayed spontaneous activity which involved large, syncronous foci, suggesting that at least a fraction of these cells were capable of self-generating action potentials. We have identified a novel type of adult stem cells, the MdPCs, as the subpopulation of cardiac vessel-derived Mabs which differentiate into a pacemaker cell-like phenotype. The cardiac differentiation potency, the expression of HCN channels underlying spontaneous activity and the sensitivity to autonomic modulation of rate are features especially suitable to a potential use of these cells as a substrate for developing implantable biological pacemakers. The major limitation of the use of mesoangioblasti is the low rate of proliferation and cardiac differentiation. One way to overcome the limitations imposed by the use of pluripotent stem cells derived from adult tissues. Two such types of cells are presently available: induced pluripotent stem (iPS) cells and pluripotent stem cells derived from testis. iPS are somatic cells reprogrammed by exogenous expression of a series of genes involved in the maintenance of ES cell pluripotency (Oct3/4, Sox2, c-Myc and Klf4 for murine cells and Oct4, Sox2, nanog and lin28 for human cells). While these cells have the advantage of not being immunogenic, they still require genetic manipulation which could potentially impair their therapeutic application. Recent data have shown that spermatogonial stem cells (SSCs), which are normally located on the basal membrane of the seminiferous tubules and are responsible for spermatogenesis, can in vitro become pluripotent cells which differentiate into derivatives of all three embryonic germ layers and form teratomas when injected in animals. We have developed a protocol of enzymatic and mechanical dissociation from wild type and transgenic mouse. The most promising protocol is the use of transgenic mouse that express the protein reporter GFP under control of trascritional factor Oct4. At the moment we obtained single cells Oct4+/EGFP+ that remain in colture until seven days but we must apply further analysis to find the optimal conditions to enable the survival and the proliferation of this cells.