Fig 1.
Proliferative behavior and viability and of pBM-MSCs co-cultured with pericardial fluid.
The pBM-MSCs were co-cultured in the presence of pericardial fluids from three different healthy animals. (A and B) The proliferative behavior of cells was spectrophotometrically quantified at 460 nm for 26 days using a CCK-8 proliferation kit. Values shown in the graphics represent mean±SD of 3 independently performed experiments. The slopes correspond to the proliferation rates of pBM-MSCs. An ANOVA test was performed comparing the slopes of the different experiments between groups, and no significant differences were found. (C and D) The percentage of viable cells was calculated by trypan blue dye-exclusion and the average percentage of live and dead cells is represented in grey and black respectively.
Table 1.
Biochemical analysis of pericardial fluid and blood plasma (n = 3).
Fig 2.
Effect of pericardial fluid on pBM-MSCs phenotype.
The phenotypic analysis was performed by multicolor flow cytometry. The cells were in vitro cultured for 7 days in the presence of Fetal Bovine Serum and in the presence of pericardial fluid at 25%, 50%, 75% and 100%. Representative histograms of four different experiments are shown (A). The expression level of Stem Cell Markers (CD29, CD31, CD44, CD90, CD105) and Swine Leukocyte Antigen Class-I and Class-II (SLA-I and SLA-II) is represented as Normalized Mean Relative Fluorescence Intensity which is calculated by dividing the Mean Fluorescent Intensity (MFI) by its isotype control. Graphic representation of mean±SD for each marker is also provided (n = 4) (B). No significant differences were found between groups when ANOVA test was performed.
Fig 3.
Optimized labeling of pBM-MSCs for MRI.
(A) Endorem was incubated with pBM-MSCs at different concentrations (25 μg/ml, 50 μg/ml, 100 μg/ml and 200 μg/ml) Superparamagnetic iron oxide particle were detected by Prussian Blue-staining and observed by optical microscopy. (B) The Prussian Blue staining was spectrophotometrically quantified on pBM-MSCs. The cells were lysed with 0.1% Triton X-100 and the absorbance of the extracts was quantified at 700 nm.
Fig 4.
In vivo cell tracking of intrapericardially delivered pBM-MSCs by cardiac-MRI in non-infarcted hearts.
A total of 100x106 SPIO-labeled pBM-MSCs cells were injected into the pericardial cavity of healthy Large White pigs. The MRI was performed using a 1.5T magnetic resonance technology. Images were acquired in four chamber views (A-D) and using a T2-star gradient echo image (E-H). Images taken at day 3 post injection (B-D, F-H) are represented together with the corresponding control images (A, E) taken before injection. The arrows indicate the location of SPIO-labeled cells.
Fig 5.
In vivo cell tracking of intrapericardially delivered pBM-MSCs by cardiac MRI in infarcted heart.
A total of 100x106 SPIO-labeled pBM-MSCs cells were injected into the pericardial cavity. The MRI was performed using 1.5T magnetic resonance technology. Images were acquired in four chambers view (A-D) and using a T2-star gradient echo image (E-H). Images taken at day 3 post injection (B-D, F-H) are represented together with the corresponding control images (A, E) taken before injection and one week after the infarction induction. The arrows indicate the location of SPIO-labeled cells.
Fig 6.
Macroscopic examination and engraftment of SPIO-labeled cells in non-infarcted heart.
The SPIO-labeled pBM-MSCs were intrapericardially injected. At 7 days, heart samples were sliced into 1–3 cm short-axis sections and were then photographed (A). The heart slices were fixed in 4% formaldehyde. For the detection of SPIO–labeled cells, tissue sections were incubated with 8% hydrochloric acid and 4% potassium ferrocyanide and then with eosin. The blue color indicates the presence of SPIO within the tissue (B). Finally, for histological examination, tissue sections were paraffin-embedded and Prussian-blue/eosin staining demonstrated a preferential distribution of SPIO–labeled cells in the left ventricular myocardium (C). Scale bar: 100μm. Black arrows indicate the presence of SPIO-labeled cells evidenced by the potassium ferrocyanide staining.
Fig 7.
Macroscopic examination and engraftment of SPIO-labeled cells in an infarcted heart.
The SPIO-labeled pBM-MSCs were intrapericardially injected one week after infarction induction. At 7 days, heart samples were sliced into 1–3 cm short-axis sections and were then photographed (A). The heart slices were fixed in 4% formaldehyde. For the detection of SPIO–labeled cells, tissue sections were incubated with 8% hydrochloric acid and 4% potassium ferrocyanide and then with eosin. The blue color indicates the presence of SPIO within the tissue (B). Finally, for histological examination, tissue sections were paraffin-embedded and Prussian-blue/eosin staining demonstrated a preferential distribution of SPIO–labeled cells in the left ventricular myocardium (C). Scale bar: 100μm. Black arrows indicate the presence of SPIO-labeled cells evidenced by the potassium ferrocyanide staining.
Fig 8.
Y chromosome detection of intrapericardially delivered pBM-MSCs.
The pBM-MSCs from a male donor were detected in the heart by Y chromosome amplification. (A) In order to determine the sensitivity of PCR amplification, pBM-MSCs from a male donor were mixed with 106 pBM-MSCs from a female donor at the indicated ratios. Genomic DNA was extracted and subjected to PCR amplification using Y chromosome specific primers. The PCR allowed the detection of male cells with a sensitivity of 100–1000 cells per 106 female cells. (B) Female pigs were intrapericardially injected with male-derived pBM-MSCs. At day 7, the animals were euthanized and heart samples were collected for PCR analysis. As negative and positive controls, the genomic DNA from female and male pBM-MSCs cells were amplified. LA = left atrium, LV = left ventricle, RA = right atrium, RV = right ventricle, PV = pericardium on the right and left ventricles, PA = pericardium on the right and left atrium.