Validation of contrast-enhanced magnetic resonance imaging to monitor regenerative efficacy after cell therapy in a porcine model of convalescent myocardial infarction

Abstract

Background: Magnetic resonance imaging (MRI) in the CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trial revealed that cardiosphere-derived cells (CDCs) decrease scar size and increase viable myocardium after myocardial infarction (MI), but MRI has not been validated as an index of regeneration after cell therapy. We tested the validity of contrast-enhanced MRI in quantifying scarred and viable myocardium after cell therapy in a porcine model of convalescent MI.

Methods and results: Yucatan minipigs underwent induction of MI and 2-3 weeks later were randomized to receive intracoronary infusion of 12.5×10(6) mismatched allogeneic CDCs or vehicle. Allogeneic CDCs induced mild local mononuclear infiltration but no systemic immunogenicity. MRI revealed that allogeneic CDCs attenuated remodeling, improved global and regional function, decreased scar size, and increased viable myocardium compared with placebo 2 months post-treatment. Extensive histological analysis validated quantitatively the MRI measurements of scar size, scar mass, and viable mass. CDCs neither altered gadolinium contrast myocardial kinetics nor induced changes in vascular density or architecture in viable and scarred myocardium. Histology demonstrated that CDCs lead to cardiomyocyte hyperplasia in the border zone, consistent with the observed stimulation of endogenous regenerative mechanisms (cardiomyocyte cycling, upregulation of endogenous progenitors, angiogenesis).

Conclusions: Contrast-enhanced MRI accurately measures scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. MRI represents a useful tool for assessing dynamic changes in the infarct and monitoring regenerative efficacy.

Keywords: adult stem cells; allogeneic transplantation; cell transplantation; magnetic resonance imaging; myocardial infarction; regeneration.

Figure 1

Study protocol. Schematic depiction of the 24-hour retention study (A) and the validation study (B).

Figure 2

Allogeneic CDCs attenuate adverse remodeling and improve global and regional function compared to controls. A,B: Matched cine short-axis images (at end-diastole [ED] and end-systole [ES]) at baseline and 2 months (videos of the cine acquisitions are provided in the Online Supplement) for a minipig treated with allogeneic CDCs (A) and a control minipig (B). Images were obtained at similar levels (note the similar morphology of papillary muscles). Changes in ejection fraction (C), end-diastolic volume (D) and end-systolic volume (E) from baseline to 2 months in controls and CDC-treated minipigs. Systolic thickening (F) and end-systolic thickness (G) in infarcted (infarct) and non-infarcted (remote) segments at 2 months post-infusion in controls and CDC-treated minipigs (* p<0.05 vs CDC-treated group).

Figure 3

Allogeneic CDCs decrease scar size, decrease scar mass and increase viable myocardial mass compared to controls, as assessed by MRI. A: Representative delayed contrast-enhanced MRI acquisitions of hearts in short-axis section at end-diastole. Scarred myocardium appears white while viable myocardium appears black. In the CDC-treated minipig the scar decreased in transmurality and viable myocardial mass increased over the period of 2 months after CDC infusion. The control minipig was characterized by scar thinning and scar expansion, with no evidence of increase in viable myocardium over the same time period. Changes in scar size (B), scar mass (C) and viable mass (D) from baseline to 2 months in controls and CDC-treated minipigs. E: Scar size at baseline and at months post-infusion in control and CDC-treated minipigs. Scar mass (F) and viable mass (G) at 2 months post-infusion in controls and CDC-treated minipigs (* p<0.05 vs CDC-treated group, # p<0.05 vs baseline).

Figure 4

Allogeneic CDCs decrease scar size and increase viable myocardial mass compared to controls, as assessed by post-mortem histology. A: Representative short-axis cardiac slices of 2 CDC-treated and 2 control minipigs at 2 months post-infusion after incubation with TTC. Slices were obtained at similar levels (note the similar morphology of papillary muscles). Viable myocardium stains brick-red, while scarred myocardium appears white (unstained). B: Representative sections from the infarcted wall of a treated and a control minipig stained with Masson's trichrome. Viable myocardium stains red, while collagenous scar stains blue. CDC-treated hearts exhibited significant amounts of viable myocardium in the infarcted wall, in the form of endocardial and epicardial muscular layers surrounding the scar and in the form of islets of viable myocardial tissue dispersed in-between the collagen fibers. In control minipigs the scar was largely transmural. Scar size (C), scar mass (D), viable mass (E) and scar transmurality (F) at 2 months post-infusion in controls and CDC-treated minipigs, as assessed by post-mortem histology (* p<0.05 vs CDC-treated group).

Figure 5

Comparison of cardiac MRI with histology for assessment of scarred and viable myocardium after cell therapy. A: TTC-stained cardiac slices matched with their corresponding delayed contrast-enhanced MRI images (from both the in vivo and ex vivo MRI datasets) for a CDC-treated and a control minipig. Areas of hyperenhancement in cardiac MRIs correspond excellently to regions of scarred myocardium in histological slices. The treated minipig MRI demonstrates endocardial and epicardial rims of non-hyperenhanced tissue in the infarcted wall which correspond excellently to endocardial and epicardial TTC-positive muscular layers surrounding the scar in histological slices. The control heart is characterized by a transmural scar, and no viable myocardium can be detected in the infarct area by either MRI or histology. Correlation of MRI measurements of scar size as % of isolated cardiac slices (for in vivo [B] and ex vivo [C] MRI), scar size as % of LV, LV scar mass and LV viable mass (D) with the corresponding histological measurements.

Figure 6

Gd-contrast myocardial kinetics in cell-treated hearts. A: A series of dynamic delayed contrast enhancement images with a fixed T1 were acquired and signal intensity of specific areas (collagenous scar, viable myocardium in the infarcted region, viable myocardium at the border zone, remote myocardium) was measured at various timepoints following contrast administration, resulting in the generation of site-specific Gd-contrast curves in all cell-treated minipigs. B: Gd-contrast kinetics pooled data from all treated minipigs. Analysis using a linear mixed effects model demonstrated that Gd-contrast kinetics were identical in cell-treated viable (non-hyperenhanced) areas and in the remote non-cell treated healthy myocardium. Image on the right in A is the corresponding histological section stained with Masson's trichrome of the inset in the MRI acquisitions.

Figure 7

Vessel density and architecture. A: Vascular density and architecture were evaluated by immunostaining for α-sarcomeric actinin, α-smooth muscle actin and isolectin. Image on the right is a high-power image of inset on left. B: Vessel density in the border zone (defined as the region at the edges of the scar comprising areas of both scarred and viable myocardium). Vessel density (C), lumen diameter (D), total wall thickness, endothelial layer thickness, smooth muscle layer thickness (E) and lumen to wall ratio (F) in viable myocardium in the infarcted wall (viable), in the collagenous scar (scar) and in the remote non-infarcted myocardium (remote) (* p<0.05 vs CDC-treated group).

Figure 8

Allogeneic CDCs attenuate myocyte hypertrophy and promote endogenous regeneration in the infarct and peri-infarct area. A: Cardiomyocyte cross-sectional area in the infarct and peri-infarct area. B: Cycling cardiomyocytes (arrows) in the infarct and peri-infarct area. C: Small TnI+ cells (arrows; image on the right is a magnification of the inset on left) in the infarct and peri-infarct area (* p<0.05 vs CDC-treated group).