c-kit+ Cardiac stem cells alleviate post-myocardial infarction left ventricular dysfunction despite poor engraftment and negligible retention in the recipient heart

Abstract

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.

Figure 1. Experimental protocol.

Left anterior coronary artery occlusion (60 min) followed by reperfusion was produced in female mice; two days later, mice received 10⁵ male murine CSCs (or PBS only) via intracoronary delivery. Heart, liver, lung, kidney, and spleen were collected at indicated time points. Each tissue was analyzed for the presence of donor CSCs by qPCR as described in Materials and Methods. In the 35-day group, hemodynamic measurements were performed prior to euthanasia to assess cardiac function.

Figure 2. Distribution and retention of CSCs in various organs of the recipient mice.

Values are means±SEM. CSCs could not be detected in liver and spleen samples at any time point examined. n = 4 for the first three time points and n = 7 for the 35 days group.

Figure 3. Intracoronary infusion of CSCs alleviates LV dysfunction induced by MI.

At 35 days, hemodynamic measurements were performed using a Millar catheter in both vehicle- (n = 9) and CSC-treated mice (n = 7). A and B, Representative pressure-volume (P-V) loops from a vehicle-treated and a CSC-treated mouse recorded at baseline and during preload manipulation by a brief occlusion of the inferior vena cava. C, D, and E, Quantitative analysis of hemodynamic variables including LV end-diastolic pressure, ejection fraction and end-systolic elastance (Ees). Values are means±SEM. *, p<0.05 vs. vehicle group.

Figure 4. Comparison of CSC retention and engraftment after intramyocardial and intracoronary delivery.

Female mice with acute MI were given 10⁵ c-kit+/lin- male murine CSCs by the intramyocardial (upper panel) or intracoronary (present study; lower panel) route. The data shown in the top panel are reproduced from our previous paper . The absolute numbers of CSCs detected in the entire heart at indicated time points are shown. The number of cells found at each time point, expressed as a percentage of the cells found at 5 min after delivery, is shown in the parenthesis. Data are means±SEM.