THREE DIMENSIONAL BLOOD AND CARDIAC STEM CELL SPHERES ENHANCE THE REGENERATIVE PERFORMANCE OF TRADITIONAL MONOLAYER CULTURE CELLS

Abstract

Combination therapy with blood and cardiac stem cells after myocardial injury provide a complimentary paracrine profile that promotes angiogenesis and the generation of new myocardium. Given evidence that culture of cardiac stem cells (CSCs) as cardiospheres enhances regenerative performance, we test the hypothesis that growing both blood and cardiac stem cells as three dimensional spheres will recapitulate a stem cell niche environment to promote cell survival and cell-mediated repair. Human left atrial appendages and blood samples were obtained from patients undergoing clinically-indicated heart surgery. After seven days of monolayer culture, equivalent numbers of circulating angiogenic cells (CACs) and CSCs were combined in suspension culture which, over 7 days of culture, spontaneously formed spherical aggregates. Immunofluorescent lineage tracking demonstrated that both CACs and CSCs were heterogeneously distributed throughout the 50-80 uM spheres. CACs and CSCs grown together as spheres proliferated slowly as compared to monolayer culture of CSCs alone (2.0±0.4 fold less, p=0.01) or monolayer culture of CACs and CSCs together (1.8±0.18 fold less, p=0.02). Media conditioned under hypoxic, low serum conditions demonstrated that spherical culture did not inhibit cytokine production of angiogenin and VEGF while providing a 1.3±0.3 fold (n=4) increase in the production of the pro-survival cytokine SDF-1α. Consistent with these findings, media conditioned by CACs and CSCs after spherical culture promoted comparable degrees of HUVEC tubule formation when compared to media conditioned by monolayer culture of CACs and CSCs together (41.1±10.6 vs. 53.4±6.5 um respectively; p=0.36) while providing markedly greater recruitment of circulating stem cells (76.6±9.3 vs. 65.7±7.8 respectively; p=0.02). Implantation of sphered CACs and CSCs one week into immunodeficient mice after LAD ligation provided greater myocardial function relative to monolayer cultured CSCs (5±4 vs. 11±1% delta LVEF, p=0.04) with hints of superior regenerative performance when compared to traditional cardiospheres (9±1% delta LVEF, p=0.09). Spherical culture of CACs and CSCs together provides an incremental benefit towards cytokine production, recruitment of circulating stem cells and cell-mediated cardiac repair. This innovative culture technique may provide a means to directly apply stem cell therapy to the clinical setting when cell product yields are severely constrained.