Embryonic stem cell-derived CD166+ precursors develop into fully functional sinoatrial-like cells

Rationale: A cell-based biological pacemaker is based on the differentiation of stem cells and the selection of a population displaying the molecular and functional properties of native sinoatrial node (SAN) cardiomyocytes. So far, such selection has been hampered by the lack of proper markers. CD166 is specifically but transiently expressed in the mouse heart tube and sinus venosus, the prospective SAN. Objective: We have explored the possibility of using CD166 expression for isolating SAN progenitors from differentiating embryonic stem cells. Methods and Results: We found that in embryonic day 10.5 mouse hearts, CD166 and HCN4, markers of the pacemaker tissue, are coexpressed. Sorting embryonic stem cells for CD166 expression at differentiation day 8 selects a population of pacemaker precursors. CD166(+) cells express high levels of genes involved in SAN development (Tbx18, Tbx3, Isl-1, Shox2) and function (Cx30.2, HCN4, HCN1, CaV1.3) and low levels of ventricular genes (Cx43, Kv4.2, HCN2, Nkx2.5). In culture, CD166(+) cells form an autorhythmic syncytium composed of cells morphologically similar to and with the electrophysiological properties of murine SAN myocytes. Isoproterenol increases (+57%) and acetylcholine decreases (-23%) the beating rate of CD166-selected cells, which express the -adrenergic and muscarinic receptors. In cocultures, CD166-selected cells are able to pace neonatal ventricular myocytes at a rate faster than their own. Furthermore, CD166(+) cells have lost pluripotency genes and do not form teratomas in vivo. Conclusions: We demonstrated for the first time the isolation of a nonteratogenic population of cardiac precursors able to mature and form a fully functional SAN-like tissue

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 \u3b1-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

Molecular composition and functional properties of f-channels in murine embryonic stem cell-derived pacemaker cells

Mouse embryonic stem cells (mESCs) differentiate into all cardiac phenotypes, and thus represent an important potential source for cardiac regenerative therapies. Here we characterize the molecular composition and functional properties of "funny" (f-) channels in mESC-derived pacemaker cells. Following differentiation, a fraction of mESC-derived myocytes exhibited action potentials characterized by a slow diastolic depolarization and expressed the I(f) current. I(f) plays an important role in the pacemaking mechanism of these cells since ivabradine (3 muM), a specific f-channel inhibitor, inhibited I(f) by about 50% and slowed rate by about 25%. Analysis of I(f) kinetics revealed the presence of two populations of cells, one expressing a fast- and one a slow-activating I(f); the two components are present both at early and late stages of differentiation and had also distinct activation curves. Immunofluorescence analysis revealed that HCN1 and HCN4 are the only isoforms of the pacemaker channel expressed in these cells. Rhythmic cells responded to beta-adrenergic and muscarinic agonists: isoproterenol (1 muM) accelerated and acetylcholine (0.1 muM) slowed spontaneous rate by about 50 and 12%, respectively. The same agonists caused quantitatively different effects on I(f): isoproterenol shifted activation curves by about 5.9 and 2.7 mV and acetylcholine by -4.0 and -2.0 mV in slow and fast I(f)-activating cells, respectively. Accordingly, beta1- and beta2-adrenergic, and M2-muscarinic receptors were detected in mESC-derived myocytes. Our data show that mESC-derived pacemaker cells functionally express proteins which underlie generation and modulation of heart rhythm, and can therefore represent a potential cell substrate for the generation of biological pacemakers

PAcemaker properties of autorhythmic cells derived from mouse embryonic stem cells

PAcemaker properties of autorhythmic cells derived from mouse embryonic stem cell

Pacemaker properties of autorhythmic cells derived from mouse embryonic stem cells

Pacemaker properties of autorhythmic cells derived from mouse embryonic stem cell

Identification of ESC-derived pacemaker myocytes by EGFP expression specifically driven by the promoter of the HCN4 gene

Identification of ESC-derived pacemaker myocytes by EGFP expression specifically driven by the promoter of the HCN4 gen