Engraftment, differentiation, and functional benefits of autologous cardiosphere-derived cells in porcine ischemic cardiomyopathy

Abstract

Background: Cardiosphere-derived cells (CDCs) isolated from human endomyocardial biopsies reduce infarct size and improve cardiac function in mice. Safety and efficacy testing in large animals is necessary for clinical translation.

Methods and results: Mesenchymal stem cells, which resemble CDCs in size and thrombogenicity, have been associated with infarction after intracoronary infusion. To maximize CDC engraftment while avoiding infarction, we optimized the infusion protocol in 19 healthy pigs. A modified cocktail of CDCs in calcium-free PBS, 100 U/mL of heparin, and 250 microg/mL of nitroglycerin eliminated infusion-related infarction. Subsequent infusion experiments in 17 pigs with postinfarct left ventricular dysfunction showed CDC doses > or =10(7) but <2.5 x 10(7) result in new myocardial tissue formation without infarction. In a pivotal randomized study, 7 infarcted pigs received 300,000 CDCs/kg (approximately 10(7) total) and 7 received placebo (vehicle alone). Cardiac magnetic resonance imaging 8 weeks later showed CDC treatment decreased relative infarct size (19.2% to 14.2% of left ventricle infarcted, P=0.01), whereas placebo did not (17.7% to 15.3%, P=0.22). End-diastolic volume increased in placebo, but not in CDC-treated animals. Hemodynamically, the rate of pressure change (dP/dt) maximum and dP/dt minimum were significantly better with CDC infusion. There was no difference between groups in the ability to induce ventricular tachycardia, nor was there any tumor or ectopic tissue formation.

Conclusions: Intracoronary delivery of CDCs in a preclinical model of postinfarct left ventricular dysfunction results in formation of new cardiac tissue, reduces relative infarct size, attenuates adverse remodeling, and improves hemodynamics. The evidence of efficacy without obvious safety concerns at 8 weeks of follow-up motivates human studies in patients after myocardial infarction and in chronic ischemic cardiomyopathy.

Figure 1

In vitro comparison of progenitor cell size and thrombogenecity. A. Mean MSC and CDC cell diameters are similar, and significantly larger than BMMCs (100 cells counted; *=p<0.0001). B. Tissue factor concentration in progenitor cell lysate from MSCs and CDCs is nearly equal, and significantly higher than BMMCs (n=3 in each group; *=p<0.001).

Figure 2

Cell aggregation and ex vivo thrombus formation. A. Microscopic examination showed no CDC clumping whether suspended in PBS with Ca²⁺ (PBS+) or without (PBS-). B. Thrombus formation 8 hours after mixture of CDCs with allogeneic blood is dependent on CDC number. C. Thrombus formation 1 hour after mixture of progenitor cells with blood is dependent on cell type and presence of Ca²⁺; the addition of heparin (100 or 200U/ml) eliminated thrombus formation (*: p<0.05 vs BMMC; †:p<0.05 vs PBS-.). D. Thrombus formation 8 hours after mixture of progenitor cells with blood is dependent on cell type; the addition of heparin abolished cell-dependent thrombus formation. The table below each bar group in panels B-D reflects composition of suspension solution; for “heparin” row: 100=100U/ml, 200=200U/ml.

Figure 2

Cell aggregation and ex vivo thrombus formation. A. Microscopic examination showed no CDC clumping whether suspended in PBS with Ca²⁺ (PBS+) or without (PBS-). B. Thrombus formation 8 hours after mixture of CDCs with allogeneic blood is dependent on CDC number. C. Thrombus formation 1 hour after mixture of progenitor cells with blood is dependent on cell type and presence of Ca²⁺; the addition of heparin (100 or 200U/ml) eliminated thrombus formation (*: p<0.05 vs BMMC; †:p<0.05 vs PBS-.). D. Thrombus formation 8 hours after mixture of progenitor cells with blood is dependent on cell type; the addition of heparin abolished cell-dependent thrombus formation. The table below each bar group in panels B-D reflects composition of suspension solution; for “heparin” row: 100=100U/ml, 200=200U/ml.

Figure 3

Short term CDC engraftment. A. Gross example of porcine heart; arrowhead marks infusion site; boxes indicate sampling areas: 1-4 are target areas; areas 5&6 are non-target. B. Luciferase activity in heart and non-target organs after infusion of 1×10⁵, 1×10⁶ or 1×10⁷ CDCs (n=2) shows virtually no off-target engraftment (numbers on Y-axis above “heart” label correspond to areas from panel A). C. Luciferase activity and troponin I (TnI) concentration after infusion of CDCs in PBS- with or without 100U/ml heparin and 250μg/ml nitroglycerin (NTG; table below Y-axis shows composition of suspension solution). Maximal engraftment (cells/g) was detected in the septum (areas 1&2). Total luciferase activity is sum of target areas and was increased by the addition of heparin. TnI concentration was significantly reduced with the addition of heparin and NTG (n=2). D. Luciferase activity and serum TnI 24 hours after cell infusion in the infarcted heart. Maximum and total luciferase activity in the target area, as well as serum TnI, were all dependent on the number of CDCs infused (n=2).

Figure 4

Long term CDC engraftment. A. Multiple mature cardiomyocytes with X-gal+ nuclei (arrows) at infarct border zone. B. X-gal+ cells in arteriole at infarct border zone (arrows). C. Immunohistochemistry revealing adjacent cardiomyocytes at infarct border zone with beta-galactosidase+ nuclei (green) and expression of α-sarcomeric actin (red), indicative of mature phenotype.

Figure 5

Measurement of relative infarct size prior to infusion and 8 weeks later showed a significant decrease in animals treated with CDCs (p=0.01, n=7), but not in the placebo group (p=0.22, n=6).

Figure 6

Eight weeks after infusion, animals treated with CDCs (n=7) had significantly greater dP/dt maximum (p=0.04, panel A) and significantly lower dP/dt minimum (p=0.03, panel B) than placebo animals (n=6).

Figure 6

Eight weeks after infusion, animals treated with CDCs (n=7) had significantly greater dP/dt maximum (p=0.04, panel A) and significantly lower dP/dt minimum (p=0.03, panel B) than placebo animals (n=6).