Differentiation Of C-kit Positive Cardiac Stem Cells Into Sinus Node-like Cells

Heart have been traditionally regarded as terminally differentiated organ that adapt to increased work and compensate for disease exclusively through hypertrophy. However, recent studies have verified the existence of resident cardiac stem cells (CSCs), which can differentiate into cardiac phenotypes representing early cardiomyocyte-like cells. However, CSCs-derived specification into sinus nodal-like cells is rarely known. Recently, we had made some exploration of cardiac stem cell's characteristics related to pacemaker cell.PartⅠ:The isolation and identification of c-kit+ cardiac stem cellsObective:For exploration of cardiac stem cell's characteristics related to pacemaker cell,we need a lot of CSCs with fine function and complete phenotype. Methods:When we established the conditions for the isolation and expansion of c-kit-positive CSCs from small explants of myocardium from atrial or ventricular specimens and from middle of hearts, the differentiation characteristics of the cells had been observed carefully. We developed a three-step procedure by culturing the cells before and after cell sorting. The purity of sorted c-kit+ cells was characterized by flow cytometry. Results:As a result of cell sorting by c-kit+ via FACS, we obtained the c-kit+ cells whose rate of purity was 22.89%. Following cell sorting, we checked the positive expression rate of CD34 and CD45 respectively in purified c-kit+ cells. The analysis indicated that the c-kit+ cell hardly expressed CD45 (0.9%) and CD34 (0.6%), which were the marker for hematopoietic stem cell and endothelial endothelial progenitor marker.The undifferentiated cells can grow as self-adherent clusters termed "cardiospheres". To identify the clonal growth pattern of c-kit+ cells, we seeded the cells at a density of 1 cell per well in growth medium. Altogether,180 single cells were deposited, and about 70% of the cells survived and began to proliferate within 5 days and about 40% of the survival cells formed spherical clusters. From the growth curves, we could see that the proliferation speed of the c-kit+ cell from cardiac apex was faster than those from atrium and middle of the heart. This proliferation speed of these cells from middle tissue of the heart was the lowest. Conclusions:Under condition of culture, these cells are clonogenic and capable of long-term self-renewal and can differentiate in vitro.PartⅡ:The identification of cardiac progenitor cells' differentiation phenotype and electrophysiological recordingsObective:The purpose of this study was to determine whether the juvenile heart of large mammals contains cardiac stem cells (CSCs), which are suitable as seed cells for restoration of cardiac pacemaker cell. Methods:The c-kit+ CSC sorted by a flow cytometry cell sorting system. Then, analyses of immunofluorescence and reverse transcription polymerase chain reaction were performed on the cells. Results:The c-kit+ CSCs we sorted were self-renewing, and they could proliferate by clonal expansion. The CSCs could differentiate into cardiac muscle, smooth muscle and endothelial cells at rates of 10.5±4.2%,13.5±5.1% and 12.9±3.5% respectively at week 4. Correspondingly, the three lineages' differentiation rates were 23.2±3.6%,25.9±6.6% and 28.3±6.1% respectively at week 8. The difference of each lineage's differentiation rate between week 4 and week 8 was of significance respectively, P< 0.05. Some cultured cells generated from the CSCs could express cardiac transcription factor GATA-4 after week 2 and express pacing-related genes, including hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) and HCN4 after week 4. The fact that a fraction of the CSCs demonstrated the presence of inward currents verified the existence of inward current channels. Conclusions:the c-kit+ CSCs may differentiate into cardiac muscle cell and sinus node-like cells, suggesting that the CSCs would be useful as seed cells in treating sinus bradycardiac disorders or exploring the mechanism of pacemaker activity. PartⅢ:Differentiation induction of Cardiac c-Kit Positive Cells from rat heart into Sinus Node-like Cells by 5-AzacytidineObective:Using the demethylating agent,5-Aazacytidine (5-Aza), we tried to follow the process of cardiac specialization into sinus node-like cells. Methods:The c-kit+ cells were isolated from one-month-old rat hearts and sorted by a flow cytometry cell sorting system. Then, analyses of immunofluorescence and reverse transcription polymerase chain reaction were performed on the cells. Results:The sorted c-kit+ cell are self-renewing and clonogenic. Some of the c-kit+ cell could spontaneously express GATA-4, cardiac troponinⅠ(cTnI), smooth muscle actin (SMA) and CD31. At week 8, the cells treated with 10μM 5-Aza expressed GATA-4 and cTnI at rates of 31.4±2.6% and 32.6±8.5%. Correspondingly, the rates were 21.4±8.1% and 18.7±4.3% in the cells without 5-Aza. The difference of GATA-4 or cTnI expression rate between two groups was of significance respectively, P< 0.05. The cultured cells could express mRNA of GATA4, hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) and HCN4. The mRNA expression was aslo up-regulated in cells treated with 10μM 5-Aza and growth factors. By week 2 after cell sorting, inward currents could be recorded in a fraction of the cultured cells. Conclusions:10μM 5-Aza could promote the differentiation of c-kit+ cells into sinus node-like cells.5-Aza-mediated differentiation seems to be helpful in future cell therapy for the patients suffering from loss of sinus node cells.