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The regenerative potential of c-kit(+) cardiac stem cells (CSCs) is severely limited by the poor survival of cells after transplantation in the infarcted heart. We have previously demonstrated that preconditioning human CSCs (hCSCs) with the heme oxygenase-1 inducer, cobalt protoporphyrin (CoPP), has significant cytoprotective effects in vitro. Here, we examined whether preconditioning hCSCs with CoPP enhances CSC survival and improves cardiac function after transplantation in a model of myocardial infarction induced by a 45-minute coronary occlusion and 35-day reperfusion in immunodeficient mice. At 30 minutes of reperfusion, CoPP-preconditioned hCSCs(GFP+), hCSCs(GFP+), or medium were injected into the border zone. Quantitative analysis with real-time qPCR for the expression of the human-specific gene HLA revealed that the number of survived hCSCs was significantly greater in the preconditioned-hCSC group at 24 hours and 7 and 35 days compared with the hCSC group. Coimmunostaining of tissue sections for both green fluorescent protein (GFP) and human nuclear antigen further confirmed greater hCSC numbers at 35 days in the preconditioned-hCSC group. At 35 days, compared with the hCSC group, the preconditioned-hCSC group exhibited increased positive and negative left ventricular (LV) dP/dt, end-systolic elastance, and anterior wall/apical strain rate (although ejection fraction was similar), reduced LV remodeling, and increased proliferation of transplanted cells and of cells apparently committed to cardiac lineage. In conclusion, CoPP-preconditioning of hCSCs enhances their survival and/or proliferation, promotes greater proliferation of cells expressing cardiac markers, and results in greater improvement in LV remodeling and in indices of cardiac function after infarction., (© 2015 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Although transplantation of c-kit+ cardiac stem cells (CSCs) has been shown to alleviate left ventricular (LV) dysfunction induced by myocardial infarction (MI), the number of exogenous CSCs remaining in the recipient heart following transplantation and their mechanism of action remain unclear. We have previously developed a highly sensitive and accurate method to quantify the absolute number of male murine CSCs in female recipient organs after transplantation. In the present study, we used this method to monitor the number of donor CSCs in the recipient heart after intracoronary infusion. Female mice underwent a 60-min coronary occlusion followed by reperfusion; 2 days later, 100,000 c-kit+/lin- syngeneic male mouse CSCs were infused intracoronarily. Only 12.7% of the male CSCs present in the heart immediately (5 min) after infusion were still present in the heart at 24 h, and their number declined rapidly thereafter. By 35 days after infusion, only ∼ 1,000 male CSCs were found in the heart. Significant numbers of male CSCs were found in the lungs and kidneys, but only in the first 24 h. The number of CSCs in the lungs increased between 5 min and 24 h after infusion, indicating recirculation of CSCs initially retained in other organs. Despite the low retention and rapid disappearance of CSCs from the recipient heart, intracoronary delivery of CSCs significantly improved LV function at 35 days (Millar catheter). These results suggest that direct differentiation of CSCs alone cannot account for the beneficial effects of CSCs on LV function; therefore, paracrine effects must be the major mechanism. The demonstration that functional improvement is dissociated from survival of transplanted cells has major implications for our understanding of cell therapy. In addition, this new quantitative method of stem cell measurement will be useful in testing approaches of enhancing CSC engraftment and survival after transplantation.