| The Purification, Culture and Differentiation of Canine Bone Marrow Mesenchymal Stem Cells in VitroObjective: To study and establish the method for isolation, culture, proliferation and identification of canine bone marrow mesenchymal stem cells (MSCs), and for committed differentiation of mesenchymal stem stem cells to Cardiomyocytes in vitro, in order to provide an ideal cell source for the restoration therapy of heart failure after myocardial infarction.Methods: Ten milliliter canine bone marrow aspirates, taken from the iliac crest of adult canines, were diluted 1:1 with RPMI1640 Medium. After layered over an equal volume of 1.074 g/ml Percoll gradient solution, centrifugated at 600 g for 30 minutes, the mononuclear cells (MNCs) were recovered from the gradient interface and washed with PBS. The bone marrow mononuclear cells were suspended in RPMI1640 medium containing 15% fetal bovine serum, 100 U/ml penicillin, 100μg /ml streptomycin. With the adherence features through isolation, culture and proliferation, MSCs were obtained and identified by special cellular surface antigens. MSCs were culture-induced for 4 weeks with different concentration of 5-azacytidine in order to committed differentiate into cardiomyocytes. The myotubes, cardiac specific antigens, cellular ultrastructure of differentiated cells were observed inmorphology, and with immunohistochemical staining and transmission electron microscope.Results The methodology for isolation and expansion of MSCs was established. The total score of the mononuclear cells was about 1.0×108. The MSCs were set very good in culture of RPMI1640 medium supplemented with 15% fetal bovine serum. MSCs attached and grew as fibroblastic cells at 24 hours after initial plating, about 10-6 mononuclear cells. MSCs grew rapidly, developed into visible symmetric colonies at about 3 days and reached confluence at 6 to 7 days. While the cells were permitted to proliferate to confluence, MSCs were spindle-shaped morphology, small and arranged like pectinate. As many as 1.1～1.5×107 cells were generated by passage 3 from a 10ml marrow aspirate. To passage 10, MSCs maintained normal mesenchymal stem cells biological features. These expanded mesenchymal stem cells were positive for SH2, and negative for CD45 surface antigen. Four weeks culturation after incubation with 6,8 or 10μmol/L 5-azacytidine, MSCs were induced to differentiate to myocyte-like cells which were positive forα-actin, and negative for troponin-â… . Transmission electron microscope showed that differentiated cells with oval nuclei positioned in the central part of the cell had myofilaments, among which 8μmol/L 5-azacytidine induced MSCs exhibited well-organized myofilaments, after culturation without 5-azacytidine, MSCs were negative forα-actin, troponin-â… , and no myofilaments under transmission electron microscope at the 4th weeks. But at the 8th weeks culturation, MSCs were positive forα-actin, and negative for troponin-â… , and transmission electron microscope showed well-organized myofilaments.Conclusions MSCs from canine bone marrow could be generated, cultured and expanded in vitro. These cells displayed a stable phenotype, biological and cellular features. MSCs from canine bone marrow could be differentiated into myogenic instead of cardiomyogenic cells by 5-azacytidine induction in vitro; but without 5-azacytidine induction, MSCs could also differentiate into myogenic cells afterlong-time (8 weeks) cultivation. The differentiated MSCs without induction would provide an ideal cell source for transplantation therapy of damaged cardiac muscle tissue.The Differentiation of Canine Autologous Bone Marrow Stem Cells Transplanted into Infracted Myocardium Objective: To observe and compare the characteristics of the growth and differentiation of canine autologous bone marrow mononuclear cells and mesenchymal stem cells which implanted into infracted myocardium by intracoronary infusion.Methods: Marrow mononuclear cells were isolated from bone marrow by ensity gradient centrifugation, then were prepared for transplantion after labeled with 5-bromo-2'-deoxyuridine (BrdU). Mesenchymal stem cells were harvested by density gradient centrifugation and adherent culture from bone marrow, then cultured, proliferated and labeled with 4', 6-Diamidino-2-phenyindole, dilactate (DAPI), then prepared for transplantation. Acute myocardial infarction model of adult cannine was made through left anterior descending branch (LAD) ligation at the middle segment. Then autologous bone marrow mononuclear cells (1.0～3.0×108, n=7) or mesenchymal stem cells (4.0～6.0×107, n=5) were infused into infarct related coronary artery (IRA) without the reperfusion of acute ischemic region, and culture medium was infused for control group (n=5). Specimens were harvested 6 weeks after the cellular transplantation for hitological study including HE, PTH, immunohistochemical stain (α-actin and Connexin-43) and transmission electromicroscope study to search implanted cells and observed their growth and differentiation.Results: The BrdU-labeling positive rate of marrow mononuclear cells in vitro was about 50% which were found in the infracted region by immunohistochemical staining with anti-BrdU monoclonal antibody six weeks after transplantation. The DAPl-labeling positive rate of marrow mesenchymal stem cells was about 100% which were found in the infracted region by fluorescence microscope and were positive stained forα-actin and Connexin-43 through immunohistochemical staining six weeks after transplantation. The implanted newborn myocytes arranged in order of consistency with host myocardial fibers, and scattered for both groups.Conclusions: Autologous bone marrow mononuclear cells and mesenchymal stem cells can both migrate into infracted region and survive six weeks after transplantion by intracoronary infusion. Marrow mesenchymal stem cells can differentiate into mature cardiomyocyte-like cells. The Therapeutic Effects of Intracoronary Transplantation of Canine Autologous Bone Marrow Stem cells after Acute Myocardial InfarctionObjects: To observe the therapeutic effects of intracoronary transplantation of canine autologous bone marrow mononuclear cells and mesenchymal stem cells after acute myocardial infarction.Methods: Bone marrow mononuclear cells were isolated from bone marrow by density gradient centrifugation, and mesenchymal stem cells were obtained from culture, proliferation in vitro after the isolation of mononuclear cells. Acute myocardial infarction (AMI) model (n=23) of adult carmine was made through left anterior descending branch (LAD) ligation at the middle segment. Then autologous bone marrow mononuclear cells (n=8), mesenchymal stem cells (n=7) or culture medium (control group, n=8) were infused into infarct related coronary artery (IRA) without the reperfusion of acute ischemic region. To evaluate the therapeutic effects of adult cannine autologous bone marrow stem cells transplantation by intracoronary infusion, we measured left ventricular functional parameters (±dp/dtmax,±dp/dtmax/LVSP and CO) through Swan-Ganz catheter monitoring, performed echocardiography to get the value of LVEF and LVEDD, at the time before LAD ligation and at 6 weeks after transplantation, determined MI area and MI size by heart TTC stain after 6 weeks, measured the number of capillary vessels (the quantity of vessels in one high power field＜0.2mm2＞on light microscope) in the infracted region.Results: 1. In control group, the MI area was 185.8±153.7mm2 and MI size was 2.77%; the number of capillary vessels was 2.03±0.46/0.2mm2 in the infracted region; the hemodynamic parameters (SBP, DBP, MBP, LVSP and LVEDP) on 6th week showed no significant difference (all p＞0.05) in comparison with those of the baseline; the parameters of left ventricular function (±dp/dtmax,±dp/dtmax/LVSP and CO) decreased significantly compared with those of the baseline; echocardiography showed that LVEF decreased significantly (35.28±4.76% vs 49.85±5.42%, p＜0.01) while other parameters (EDV, ESV, SV and LVEDD) were not significantly different with those of the baseline(p＞0.05).2. Compared with the control group, the MI area and MI size in mononuclear cell group decreased 30.9% and 35.0%, respectively, without significance (p＞0.05) though; the number of capillary vessels in the infracted region increased significantly to 5.33±0.58 (p＜0.01); the hemodynamic parameters (SBP, DBP, MBP, LVSP and LVEDP) on 6th week showed no significant difference (all p＞0.05); the parameters of left ventricular function (±dp/dtmax,±dp/dtmax/LVSP and CO) increased significantly (p＜0.05～0.01); echocardiography showed that LVEF increased significantly (46.1±7.71%, p＜0.01) and other parameters (EDV, ESV, SV and LVEDD) were not significantly different (p＞0.05).3. Compared with the control group, the MI area and MI size in mesenchymal stem cell group decreased 45.9% and 48.0%, respectively, without significance (p＞0.05) though; the number of capillary vessels in the infracted region increased significantly to 5.49±0.50 (p＜0.01); the hemodynamic parameters (SBP, DBP, MBP, LVSP and LVEDP) on 6th week showed no significant difference (p＞0.05); the parameters of left ventricular function (±dp/dtmax,±dp/dtmax/LVSP and CO) increased significantly (p＜0.05～0.01); echocardiography showed that LVEF increased significantly (48.25±8.57%, p＜0.01) and other parameters (EDV, ESV, SV and LVEDD) were not significantly different (p＞0.05).4. The parameters observed in this study showed no significant difference between mononuclear cell group and mesenchymal stem cell group (p＞0.05).Conclusions: The intracoronary transplantation of autologous bone marrow mononuclear cells and mesenchymal stem cells can enhance neoangiogenesis, decrease the MI size, improve the systolic and diastolic function after AMI. The therapeutic effects of both stem cell are similar.
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