This study was reviewed and approved by the University of Miami Institutional Animal Care and Use Committee and complies with all Federal and State guidelines concerning the use of animals in research and teaching as defined by The Guide For the Care and Use of Laboratory Animals (NIH Pub. No. 80-23, revised 1985).
For this study, 31 healthy female Yorkshire swine weighing 25-35 kg underwent experimental myocardial infarction (MI) followed by reperfusion [Online. Figure 1
]. The study was conducted in 2 phases. In the first phase, three groups were studied: Yorkshire pigs received transendocardial injections (TEI) (Stiletto, Boston Scientific, Natick, Massachusetts) of 75×106
GFP labeled MSCs (n=8), Placebo (n=8) or no injection (n=3) three days following the MI. Animals were sacrificed at 24h (n=2 placebo and n=2 MSCs treated), 72h (n=3 placebo and n=3 MSCs treated) and 2 weeks (n=3 MSCs treated n=3 placebo and n=3 control) after transplantation in order to study the fate of the allogeneic cells.
In the second phase, two groups were studied: The Yorkshire pigs were randomized to receive TEI of 100×106 cells of male GFP-labeled MSCs or their 10x concentrated condition medium 3 days after MI and followed by MRI analyses (Siemens Symphony, Erlangen, Germany) at multiple time points (baseline, 2 days post MI, 4days, 2 weeks and 8 weeks post-injection)in order to assess the amount of functional recovery. The animals were sacrificed at 2 weeks (n=3 CCM and n=3 MSCs treated) and 8 weeks (n=3 CCM and n=3 MSCs treated) after injections.
GFP transduction of MSCs
Passage 1 (P1) MSCs were plated in a T25cm2 flask and transduced with lentiviral green fluorescence protein (GFP). At approximately 50% confluency the media was removed and replaced with 5ml of transduction media consisting of alpha MEM plus 20% FCS plus 8 ug/ml polybrene and 10 ul of lenti viral vector LV-173GFP (Lentigen, Gaithersburg, MD). The culture flask was incubated for 72 hours total, with fresh transduction media being added every 24h. The next day the media was removed and alpha MEM plus 20%FCS added to the culture. The flask was further incubated until confluent.
Cultures were expanded with each passage of the GFP+ MSC until sufficient numbers of MSC were obtained. The cells were then frozen in liquid nitrogen until needed. Prior to injection, the cells were thawed rapidly and washed to remove dimethylsulfoxide (DMSO), then resuspended in PBS plus 1% human serum albumin (HSA) to the required cell dose.
Transendocardial Injections of MSCs
Delivery of the cells at the sites of myocardial injury was performed as previously described22
. Briefly, left ventriculograms from 2 different angiographic projections [left (-30°) and right (+30°) anterior oblique] were used to manually map the endocardial contours of the LVs in both projections. The IZ and BZ were then delineated on the contours, and a total of 15 injections were performed in each animal, with each injection containing 0.5 ml of the injectate. Each injection was fluoroscopically guided to distribute cells evenly throughout the entire infarct and border zones.
For microscopic evaluation, the regions of interest were selected from each of the transverse ventricular sections based on CMRI and gross pathology findings: (i). One sample from the middle of the scarred, infarcted tissue. (ii) One sample containing the left border of the infarct along with non-scarred tissue; one sample containing the right border of the infarct along with non-scarred tissue (these two samples were defined as the border zones macroscopically). Microscopically, within these samples, border zones were defined as the areas that were 1-1 ½ high power fields distant from scarred zones. (iii) One sample from the posterior non-infarcted LV wall. Confocal analysis was performed as previously described.
All the values are presented as means ± SEM. All analyses were performed by using the SPSS for Windows version 15.0 (SPSS Inc., Chicago, IL). Differences between groups following immunohistological evaluation were compared by using One Way ANOVA. Differences between groups in ejection fraction and infarct size based on cMRI were calculated by using two-way repeated measures ANOVA. The Tukey's test was used for the post-hoc analysis. A level of P≤0.05 was considered statistically significant.
A detailed Materials and Methods section describing all procedures and protocols is provided in the online supplement