Dose-dependent improvement of cardiac function in a swine model of acute myocardial infarction after intracoronary administration of allogeneic heart-derived cells

Abstract

Background: Allogeneic cardiac-derived progenitor cells (CPC) without immunosuppression could provide an effective ancillary therapy to improve cardiac function in reperfused myocardial infarction. We set out to perform a comprehensive preclinical feasibility and safety evaluation of porcine CPC (pCPC) in the infarcted porcine model, analyzing biodistribution and mid-term efficacy, as well as safety in healthy non-infarcted swine.

Methods: The expression profile of several pCPC isolates was compared with humans using both FACS and RT-qPCR. ELISA was used to compare the functional secretome. One week after infarction, female swine received an intracoronary (IC) infusion of vehicle (CON), 25 × 106 pCPC (25 M), or 50 × 106 pCPC (50 M). Animals were followed up for 10 weeks using serial cardiac magnetic resonance imaging to assess functional and structural remodeling (left ventricular ejection fraction (LVEF), systolic and diastolic volumes, and myocardial salvage index). Statistical comparisons were performed using Kruskal-Wallis and Mann-Whitney U tests. Biodistribution analysis of 18F-FDG-labeled pCPC was also performed 4 h after infarction in a different subset of animals.

Results: Phenotypic and functional characterization of pCPC revealed a gene expression profile comparable to their human counterparts as well as preliminary functional equivalence. Left ventricular functional and structural remodeling showed significantly increased LVEF 10 weeks after IC administration of 50 M pCPC, associated to the recovery of left ventricular volumes that returned to pre-infarction values (LVEF at 10 weeks was 42.1 ± 10.0% in CON, 46.5 ± 7.4% in 25 M, and 50.2 ± 4.9% in 50 M, p < 0.05). Infarct remodeling was also improved following pCPC infusion with a significantly higher myocardial salvage index in both treated groups (0.35 ± 0.20 in CON; 0.61 ± 0.20, p = 0.04, in 25 M; and 0.63 ± 0.17, p = 0.01, in 50 M). Biodistribution studies demonstrated cardiac tropism 4 h after IC administration, with substantial myocardial retention of pCPC-associated tracer activity (18% of labeled cells in the heart), and no obstruction of coronary flow, indicating their suitability as a cell therapy product.

Conclusions: IC administration of allogeneic pCPC at 1 week after acute myocardial infarction is feasible, safe, and associated with marked structural and functional benefit. The robust cardiac tropism of pCPC and the paracrine effects on left ventricle post-infarction remodeling established the preclinical bases for the CAREMI clinical trial (NCT02439398).

Keywords: Acute myocardial infarction; Allogeneic; CPC; Cardiac progenitor/stem cells; Intracoronary administration; Swine model.

Conflict of interest statement

JLA, BS, RR, VA, LRB, and IP were employees of Coretherapix (part of the Tigenix Group since July 2015). The remaining authors declare that they have no competing interests.

Figures

Fig. 1

Large animal studies. Experimental workflow. a Flow chart illustrating the study design in large white swine. b AMI induction, treatment and sacrifice timetable, AMI indicates acute myocardial infarction. pCPC, cardiac stem/progenitor cells isolated from large white swine. LAD, left anterior descending coronary artery; CMR, cardiac magnetic resonance; PET, positron emission tomography

Fig. 2

Phenotypic and functional characterization of pCPC. Comparison with hCPC. a Swine CPC characterization by flow cytometry. Expression of CD90, CD105, CD45, SLAI, SLAII, and CD86 is shown (empty histogram) and the number of positive cells is indicated (%). Gray-filled area represents isotype control. b RT-qPCR analysis of PECAM1 (CD31), GATA4, and GATA6 expression in the pCPC batches. Ct value for each sample/gene analyzed. There are no significant differences between the batches used. The average expression normalized to beta-2-microglobulin (β2M) is shown. Error bars represent SD (n = 3). c Comparative expression analysis of F11R and CACNG7 membrane makers, in both swine and human isolates; three independent isolates were compared for each cell type. The assay was performed three times, and data are expressed as mean ± SD; black lines indicate the p value summary (***< 0.002, **< 0.02, *< 0.05) (one-way analysis of variance followed by the Bonferroni multiple comparison test). d Porcine CPC (n = 4) secretome characterization by ELISA compared to human CPC (n = 3) secretome. The results are expressed as mean ± SD in pg/mL. e Migration assay. Conditioned medium (CM) of human cells (CPC1, CPC3, MSC, and HDF), were compared with CM obtained from swine samples (pCPC3 and pCPC5, pMSC and IPAM (pig alveolar macrophages) in their capacity to trigger the migration of MonMac-1 cells

Fig. 3

Acute toxicity and biodistribution of pCPC in infarcted swine. a cTnI values measured over the course of the study. The slight elevation seen 24 h after vehicle/cell administration was not significantly different between groups. b Coronariogram obtained immediately after pCPC administration in a 50-M animal depicting complete opacification of the artery (TIMI 3). c Cytokine levels measured in plasma samples. Bars show the differences at 24 h after vehicle/cell administration referred to pre-administration values. d PET/CT images after 18F-FDG-labeled CPC administration. pCPC labeled with 18F-FDG were intracoronary administered in pigs 1 week after infarction. Cell distribution was analyzed by PET 4 h after cell infusion. d PET maximal intensity projection (MIP) images, showing the distribution of 18F-FDG activity over the entire body of the animal. e18F-FDG activity could also be clearly detected in the bladder (b), kidneys (k), and lungs (l). f Sagittal sections of PET/CT images only in the heart area; a diffuse uptake is shown

Fig. 4

Evaluation of early edema 1 week after the treatment. Mean edema percentage before and 1 week after pCPC administration in animals receiving (a) vehicle (CON), (b) 25 × 106 pCPC (25 M), or (c) 50 × 106 pCPC (50 M). d Short-axis images of a mid-ventricular slice acquired pre-injection and f 1 week post-injection in a representative animal belonging to the 50 M group. p values obtained using non-parametric tests (Mann-Whitney U test)

Fig. 5

Evolution of myocardial damage parameters after pCPC administration in infarcted swine. a–c Changes over time in cardiac function parameters as measured with cardiac magnetic resonance (CMR) for the three experimental groups (CON, vehicle; 25 M, receiving 25 × 106 pCPC; 50 M, receiving 50 × 106 pCPC). Treatment effects (defined as the difference between pre-injection and 10-week values). a Changes in left ventricular ejection fraction (LVEF). b End-diastolic volume indexed to body surface area (EDVi). c End-systolic volume indexed to body surface area (ESVi). d Representative CMR and TTC-stained slices from the three studied groups. e Myocardial edema/area at risk (AAR) was calculated as a percentage of the left ventricle in a mid-heart slice using T2-weighted imaging. f Final infarct size (FIS) in an equivalent slice. g Myocardial salvage index (MSI) was then computed as AAR at mid-heart slice minus FIS in an equivalent slice divided by AAR (MSI = (AAR-FIS)/AAR). p values obtained using non-parametric tests (Kruskal-Wallis and Mann-Whitney U tests)

Fig. 6

Histopathological studies. a Hematoxilin-eosin and Massons trichromic stains show typical histological appearance of the infarcts in control animals with increased collagen, while viable myocardial muscle bundles can be seen in treated animals. The bar represents 500 μm. b, c Distribution of vessels’ sizes, as determined at the infarct border

Fig. 7

Feasibility and safety study in healthy swine. pCPC (35 × 106) were administered via the LAD in healthy swine (n = 7). a Changes to cTnI (μg/L) observed at 4 h (T1) and 24 h (T2) after injection. b DE-CMR obtained at 24 h and 7 days showed no evidence of infarction. Representative short-axis image obtained 24 h after injection. c Hematoxilin-eosin staining of pigs hearts 3 weeks after cell administration. No tissue alterations or inflammatory processes were found in any case