Abstract 19874: CCN2/CTGF as a Novel Stimulator of Proliferation and Survival of Cardiac Progenitor Cells/ Stem Cells.
Background: Stem cell therapy is a novel tentative treatment option for myocardial infarction. Current challenges are more effective means of proliferation and survival of cardiac stem cells, as well as development of procedures for transplantation and engraftment of cardiac stem cells in the heart. In this study we investigate the function of the matricellular protein CCN2 on stem cell proliferation and survival.
Methods: Cardiospheres were isolated from mouse myocardial tissue, propagated and maintained in IMDM/CEM media designed to stimulate formation of cardiospheres and proliferation of cardiac progenitor cells/stem cells.(Messina et al.CircRes. 2004;95:911–921.) Immunocytochemical analysis confirmed the phenotypic characteristic of cardiac stem cells positive for Sca-1, C-kit, Gata4, Nkx−2.5 and Isl−1. Cell proliferation was analyzed by EdU Flow Cytometry. In addition, cardiac progenitor cells exposed to H2O2 damage (100 μmol/L) was compared in the presence or absence of CCN2 (300nmol/L). Recombinant human CCN2 was purified from the cell culture media of HEK293 cells and purified by sequential affinity chromatography (heparin-Sepharose) and ion-exchange chromatography (S-Sepharose) to more than 95% purity.
Results: Recombinant human CCN2 stimulated cardiosphere formation and cardiosphere growth. CCN2 stimulated proliferation of cardiac stem cell by stimulating progression through S and G2 phase of the cell cycle. CCN2 stimulated cell cycle progression was concentration dependent and correlated with AKT/GSK-3β phosphorylation (EC50 ∼ 250 nmol/L). Cell cycle progression was shown to be dependent on AKT/GSK-3β signaling (abrogated by PI3 kinase inhibitor LY294002 and AKT inhibitor API-2) and was associated with robust upregulation of cyclin D2 mRNA expression in cardiac stem cells. The survival of cardiac progenitor cells was significantly increased in the presence of CCN2 as assayed by activity of cellular reductases (MTT Survival Assay).
Conclusion: In the current study we demonstrate that CCN2 stimulate proliferation and survival of cardiac stem cells by activation of AKT/GSK-3β signaling and upregulation of cyclin D2. CCN2 may become an important factor for amplification and survival of cardiac stem cells in stem cell therapy
- © 2010 by American Heart Association, Inc.