Intrapericardial Delivery of Cardiosphere-Derived Cells: An Immunological Study in a Clinically Relevant Large Animal Model

Abstract

Introduction: The intrapericardial delivery has been defined as an efficient method for pharmacological agent delivery. Here we hypothesize that intrapericardial administration of cardiosphere-derived cells (CDCs) may have an immunomodulatory effect providing an optimal microenvironment for promoting cardiac repair. To our knowledge, this is the first report studying the effects of CDCs for myocardial repair using the intrapericardial delivery route.

Material and methods: CDCs lines were isolated, expanded and characterized by flow cytometry and PCR. Their differentiation ability was determined using specific culture media and differential staining. 300,000 CDCs/kg were injected into the pericardial space of a swine myocardial infarcted model. Magnetic resonance imaging, biochemical analysis of pericardial fluid and plasma, cytokine measurements and flow cytometry analysis were performed.

Results: Our results showed that, phenotype and differentiation behavior of porcine CDCs were equivalent to previously described CDCs. Moreover, the intrapericardial administration of CDCs fulfilled the safety aspects as non-adverse effects were reported. Finally, the phenotypes of resident lymphocytes and TH1 cytokines in the pericardial fluid were significantly altered after CDCs administration.

Conclusions: The pericardial fluid could be considered as a safe and optimal vehicle for CDCs administration. The observed changes in the studied immunological parameters could exert a modulation in the inflammatory environment of infarcted hearts, indirectly benefiting the endogenous cardiac repair.

Fig 1. Experimental design.

Seven weeks after infarct model creation, CDCs were intrapericardially injected. 30 days after CDCs administration, animals were euthanized. MRI was performed on days 0 (before CDCs administration), 7 and 30. On days 0 and 30, blood and pericardial fluid samples were collected for flow cytometry, biochemical analysis and cytokine determinations.

Fig 2. Characterization of cardiosphere-derived cells.

CDCs were isolated from cardiac tissue explants of healthy pigs. The figure A shows explants in culture with some fibroblast-like cells migrating from them (A.I), cardiospheres with CDCs migrating from them (A.II) and CDCs in culture (A.III). Figure B shows the phenotypic analysis of CDCs by flow cytometry. Representative histograms together with the expression levels are shown. The expression level of cell surface markers is represented as Mean Relative Fluorescence Intensity (MRFI), which is calculated by dividing the Mean Fluorescent Intensity (MFI) (black lined histogram) by its negative control (grey lined histogram). Figure C corresponds to gene expression analysis by conventional RT-PCR. Mean ± SD of three different experiments are shown. Data are expressed as expression percentage referred to ACTB, used as control. The relative quantification was made by measuring the brightness intensity of each band with GeneSnap software. A representative image of one of the experiments is shown above. Figure D shows the differentiation potential of CDCs. Cells were maintained for 21 days with standard medium (control) (D.I) or with specific differentiation media for adipogenic, chondrogenic and osteogenic lineages. Differentiation was evidenced by specific stainings: Oil Red O for adipocytes (D.II), Alcian Blue for chondrocytes (D.III) and Alizarin Red S for osteocytes (D.IV).

Fig 3. Lymphocyte subsets distribution in peripheral blood and pericardial fluid.

Pericardial fluid lymphocytes (PFLs) and peripheral blood lymphocytes (PBLs) were collected before CDCs administration and 30 days post-administration for flow cytometry analysis. * Statistically significant differences (p<0.05) between different time points (n = 4). † Statistically significant differences (p<0.05) between PFLs and PBLs in the same time point (n = 4).

Fig 4. Cytokines levels in pericardial fluid and plasma samples.

Cytokines levels were determined before CDCs administration and 30 days post-administration using the Luminex xMAP technology. * Statistically significant differences (p<0.05) between different time points (n = 4). † Statistically significant differences (p<0.05) between PF and plasma in the same time point (n = 4).

Fig 5. Cardiac magnetic resonance imaging.

Cardiac function was measured with cardiac magnetic resonance imaging. The panel A represent a representative image of the measurement of the thickness of the infarcted (septum) and healthy lateral free wall in end diastolic short axis views. B and C graphics represent the infarct area and the ejection fraction measurements, respectively, obtained on days 0 (before CDCs administration), 7 and 30. The lower boundary of the box indicates the 25th percentile and the upper boundary the 75th percentile. Bars above and below the box indicate the 90th and 10th percentiles. The line within the box marks the median. No statistically significant differences were found between groups (n = 4).