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Cardiac stem cells (CSCs) are promising candidates for use in myocardial regenerative therapy. We test the hypothesis that growing cardiac‐derived cells as three‐dimensional cardiospheres may recapitulate a stem cell niche‐like microenvironment, favoring cell survival and enhancing functional benefit after transplantation into the injured heart. CSCs and supporting cells from human endomyocardial biopsies were grown as cardiospheres and compared with cells cultured under traditional monolayer condition or dissociated from cardiospheres. Cardiospheres self‐assembled into stem cell niche‐like structures in vitro in suspension culture, while exhibiting greater proportions of c‐kit +  cells and upregulated expression of SOX2 and Nanog. Pathway‐focused polymerase chain reaction (PCR) array, quantitative real‐time PCR, and immunostaining revealed enhanced expression of stem cell‐relevant factors and adhesion/extracellular‐matrix molecules (ECM) in cardiospheres including IGF‐1, histone deacetylase 2 (HDAC2), Tert, integrin‐α 2 , laminin‐β 1 , and matrix metalloproteinases (MMPs). Implantation of cardiospheres in severe combined immunodeficiency (SCID) mouse hearts with acute infarction disproportionately improved cell engraftment and myocardial function, relative to monolayer‐cultured cells. Dissociation of cardiospheres into single cells decreased the expression of ECM and adhesion molecules and undermined resistance to oxidative stress, negating the improved cell engraftment and functional benefit in vivo. Growth of cardiac‐derived cells as cardiospheres mimics stem cell niche properties with enhanced “stemness” and expression of ECM and adhesion molecules. These changes underlie an increase in cell survival and more potent augmentation of global function following implantation into the infarcted heart. STEM CELLS 2010;28:2088–2098

stem cells

Cardiospheres Recapitulate a Niche‐Like Microenvironment Rich in Stemness and Cell‐Matrix Interactions, Rationalizing Their Enhanced Functional Potency for Myocardial Repair