Primary And Repeated Passage Of Bone Marrow Mesenchymal Stem Cell Transplantation In The Treatment Of Acute Myocardial Infarction, The Role Of A Comparative Study

Aims: To develop an effective therapeutic strategy for cardiac tissue regeneration by the transplantation of bone marrow mesenchymal stem cells (BM-MSCs), we compared therapeutic effects of the primary mouse BM-MSCs and the 5th passage BM-MSCs in a mouse model of acute myocardial infarction (MI) and investigated mechanisms underlying repeted passage-related effects by compared the biological characterizations of the primary mouse BM-MSCs and the 5th passage BM-MSCs in vitro. Meanwhile, we search for the way of notably amplifying BM-MSCs and overcome the limitation of amplification through repeated passage.Methods: Left ventricular anterior transmural MI model was established by permanent ligation of theramus descendens anterior arteriae coronariae sinistrae with silk ligature using male C57BL/6J wild type mice. Primary and passage 5 BM-MSCs derived from theβ-galactosidase transgenic mice by the way of"Pour-off"and repeated passage cultures were intramyocardially transplanted into the mouse MI model. 6 weeks later, animals/myocardium tissues from primary BM-MSCs group (MI+1st BM-MSCs), passage 5 BM-MSCs group (MI+5th BM-MSCs), MI group (MI) and sham group (Sham) were examined using echocardiography and histopathology or immunoblotting. Meanwhile, Primary and passage 5 BM-MSCs derived fromβ-galactosidase transgenic mice were cultured by way of"Pour-off"and repeated passage induced towards cardiomocytes and vascular endothelial cells in vitro. And expressions of lineage specific markers of cardiomocytes and vascμlar endothelial cells by those cells were examined using Real-time RT-PCR. The proliferation and apoptosis between Primary and passage 5 BM-MSCs were detected by Flow cytometry after PI stain. Meanwhile, expressions of markers of pluripotent stem cells, committed stem cells about myocardium and endothelial tissues by primary and passage 5 BM-MSCs and matrix metalloproteinases (MMP3/MMP9/MT1-MMP) were examined using Real-time RT-PCR. Furthermore, total protein lysates prepared from primary and passage 5 BM-MSCs were analyzed using Mass spectrometry (MS) following Two-dimensional (2-D) electrophoresis.Results: 6 weeks later, in comparison to passage 5 BM-MSCs, primary BM-MSCs had better therapeutic effects in the mouse MI model: readily being engrafted into the scarred myocardium and border zone, proliferating gradually in situ, forming mature vessels and cardiomocytes, promoting angiopoiesis, reversing wall thinning in scarred areas and improving cardiac functions. Moreover, levels of Akt, phospho-Akt, phospho-STAT5 detected in lysate samples prepared from primary BM-MSCs transplanted hearts were higher than in those prepared from passage 5 BM-MSCs transplanted hearts. However, levels of Pro-Caspase 3 and Activate-Caspase 3 in lysate samples prepared from primary BM-MSCs transplanted hearts were lower than in those prepared from passage 5 BM-MSCs transplanted hearts. Primary BM-MSCs maintained greater differentiation potentials as indicated by higher expressions of cardiomyocyte and vascular endothelial cell mature markers in vitro. In comparation to passage 5 BM-MSCs, primary BM-MSCs had better potential of priliferation and were hard to be apoptosis. And gene expressions of markers for pluripotent stem cells, cardiac committed stem cells, endothelium committed stem cells and matrix metalloproteinases were also expressed higher by primary BM-MSCs than those expressed by passage 5 BM-MSCs. Interestingly, 27 proteins were identified by 2-D gels and ImageMaster 2D Platinum 6.0 to be differentially expressed for 5 times by primary BM-MSCs, comparable to the passage 5 BM-MSCs. Among those proteins, 24 proteins were down-regulated and 3 proteins were up-regulated in passage 5 BM-MSCs. Discovered by biology informatics analyse and the past papers, altered proteins expression profile between primary BM-MSCs and the 5th passage BM-MSCs may affect multiple cellular functions in the repeat passage cells, including enhancing adhesion, homing, engraftment, proliferation, differentiation potential, angiogenesis, anti-apoptosis/oxidative stress, or modulation of inflammatory and immune responses. Primary BM-MSCs may be superior to passage 5 BM-MSCs in the above cellular functions.Conclusion: The maintenance of stemness in primary BM-MSCs is better than that in passage 5 BM-MSCs. The transplantation of primary BM-MSCs is superior to that of passage 5 BM-MSCs for the mouse MI model. Our data indicate that transplantation of primary BM-MSCs by way of"Pour-off"may be a more effective therapeutic strategy for cardiac tissue regeneration following MI.