Most clinical trials of autologous bone marrow-derived cell therapy in cardiovascular disease have employed the route of intracoronary injection using a balloon/catheter device and/or using G-CSF to stimulate the mobilization of bone marrow cells into the circulation (2
). These methods have several limitations. Large portions of the intracoronary injected cells flow into the blood stream, and only a small number of the cells actually migrate into the ischemic myocardium. The majority of non-expanded mononuclear cells from the bone marrow are hematopoietic lineage cells. The intracoronary injection is only applicable for non-ex vivo expanded cells (or after short expansion times of 4 days or less), since the cell size of expanded cells are much bigger than non-expanded cells (10–15 vs. 5 μm in diameter. This will dramatically limit their benefits. The direct injection of expanded MSC used in this study facilitates the ability to determine both the beneficial effects and the fate of the injected cells.
The optimal way to perform cell therapy is to allogenically transplant cells to recipients, or use a developed cell line for all recipients, since theoretically such stem cell lines would lack MHC1 and would not incite rejection. However, there is no solid evidence to prove that is always the case. The clinical trials of cell-based therapy in cardiovascular diseases are still limited to autologous use of the patients’ own bone marrow cells (2
). Similarly in order to avoid possible rejection related issues, we chose to use autologous cells in this study. In addition, we carefully tested the karyotype of the cells before injection to ensure the ex-vivo expansion did not result in any transformations or translocations of the chromosomes, which has been reported to happen in mouse and human bone marrow-derived cells following ex-vivo expansion (19
In preparing the cells used in this study, we chose to use two methods used by other investigators for MSC and endothelial progenitor cells (22
). We found that cells cultured by either method share the same characteristics of morphology and cell surface markers. We also found that they are able to differentiate into adipocytes or osteocytes, except that the growth rate is faster in EGM2 than in DMEM. The cells cultured in both conditions are classified as MSC according to the published guidelines for the nomenclature of the stem cells (24
). Furthermore in the time course study, we injected cells from both culture conditions separately and saw no difference in their differentiation.
After cell injection, we found that the cells survived in the myocardial environment, and differentiated into vascular cells, smooth muscle or endothelial cells. We certainly cannot exclude the possibility that those new regenerated cells may be the result of the paracrine effect of the injected cells causing recruitment from other sources. However, the histological findings in our study strongly suggest that these grouped vessels arose directly from the injected cells, because a paracrine effect would likely lead to a more diffuse distribution of cells. In our study, we did not see any evidence that the injected MSC differentiated into functional or morphologically analogous cardiac myocytes.
The functional tests we used for this study are echocardiography for wall motion and MRI for ejection fraction. Both analysis showed statistically significant improvements in cell treated animals at six weeks follow up compared with baseline. No such functional improvement was seen in the saline injected control animals. While the average baseline wall thickness in control animals was higher than in the cell injected animals this difference was not significant (p = 0.06). Additionally the baseline difference cannot explain the difference noted at follow up due to the fact that all animals were randomly divided before treatment and data was recorded and analyzed by operators blinded to the treatment that the animals received. Finally, the statistical analysis also showed no significant difference in baseline wall thickness between the two groups both in untreated normal areas as well.
Shake et al (25
) reported functional improvement results in a porcine model of acute myocardial infarction using mesenchymal stem cells. Their functional analysis showed preservation of function, albeit without significant improvement, after an infarct was treated with MSC. Our work attempted to mimic the clinical scenario and evaluated the effect MSC has on chronically ischemic myocardium using clinically employed methods of assessment; dobutamine stress echocardiography and cine MRI. Similar to the previous report (27) we also found that mesenchymal stem cells engraft into host myocardium when implanted by direct injection and MSC expressed muscle-specific proteins. Our data further confirmed that in various time points after injection, the cells survived and produced evidence of angiogenesis. The most interesting findings of our study are that we tracked several injection channels which contained CD90 positive and CD68 and 163 negative cells and the clusters of newly formed immature vessels lined with endothelial cells and irregular smooth muscle cells. We also demonstrated that there is no myocardial regeneration incited by MSC transplantation.