Experimental Study On Transplantation Of Mesenchymal Stem Cells With Multiple Intravenous Transplantation For Chronic Ischemic Heart Disease

Ischemic heart disease(IHD) is one of the major killers of human health, which with a high mortality and disability. Although the majority of patients can benefit from percutaneous transluminal coronary angioplasty(PTCA)、coronary artery bypass graft(CABG) and drug treatment, there are still quite a number of chronic ischemic heart disease(CHD) patients cannot benefit from cardiac intervention or surgical operation because of severe or diffused occlusion of coronary arteries. And in most cases, they also can’t benefit from drug treatment. To date, those CHD patients are lack of effective interventions, which also called “no option” patients.However, advances in stem cell research has brought hope for these patients. In recent years, a large number of experimental and clinical findings have suggested that stem cell therapy could reduce apoptosis of myocardial cells, promote angiogenesis,improve ventricular remodeling, improve heart function, which showed a good prospect. Mesenchymal stem cells(MSCs) is widely used by researchers for its wide source, simple operation, immune exemption and other advantages. Many researches have demonstrated the safety and efficiency of stem cell therapy in acute ischemic heart disease, but there is limited study in the treatment of CHD. However, there are as yet no data about the effect of MSCs delivered by combined administration on chronic myocardial ischemia in large animal model at home and abroad.MSCs has a variety of sources, while bone marrow-derived mesenchymal stem cells(BM-MSCs) was widely used in the recent studies. However, BM-MSCs has the disadvantage of the invasive process of bone marrow aspiration and the limited proliferation ability of cells from old donors. Therefore, researchers are exploring new sources of MSCs. Umbilical cord-derived mesenchymal stem cells(UC-MSCs) is thought to the ideal cell sources as an alternative to BM-MSCs because of(a) absence of invasiveness,(b)no ethical restrictions,(c)the short doubling time and potential for transdifferentiation into various cell lineages,(d)can secrete a variety of angiogenic factors and so on. At present, UC-MSCs has been used to treat a variety of diseases in pre clinical or clinical studies, including cirrhosis, diabetes, systemic lupus erythematosus, graft-versus-host disease(GVHD), etc, but in the study of heart diseases it is still in the initial stage. UC-MSCs for the treatment of CHD has not been reported yet.Therefore, this study intends to establish the animal model of CHD in pigs firstly,then intracoronary administration combined with multiple intravenous infusions of pig bone marrow-derived mesenchymal stem cells(p BM-MSCs) and human umbilical cord-derived mesenchymal stem cells(h UC-MSCs) to intervene. Finally,we used the coronary angiography, echocardiography, single-photon emission computed tomography(SPECT) and histopathological methods to evaluate the therapeutic effect of bone marrow and umbilical cord derived MSCs transplantation for CHD, which will provide theoretical basis for the clinical application in the future.Firstly, p BM-MSCs were isolated by Ficoll density gradient centrifugation method. They were spindle-shaped, spiral or fishtail growth. Flow cytometric analysis revealed that they were positive for CD29, CD44, CD90, and negative for CD14,CD34, CD45, CD166, HLA-DR. Furthermore, they were able to differentiate into osteogenic, adipogenic and chondrogenic lineages under specific conditions in vitro.Secondly, h UC-MSCs were collected through enzyme digestion, they were spindle-shaped, vortex or parallel arrangement. Flow cytometric analysis revealed that they were positive for CD29, CD44, CD73, CD90, CD105, HLA-ABC, and negative for CD34, CD45, HLA-DR. Besides, h UC-MSCs also have the potential to differentiate into osteogenic, adipogenic and chondrogenic lineages in vitro.Finally, we discuss the therapeutic effect of p BM-MSCs and h UC-MSCs on the large animal model of CHD. An Ameroid constrictor was placed around the left circumflex coronary artery(LCX) in a total of 30 miniature pigs to establish the animal model of CHD. Four weeks later, coronary angiography and electrocardiogram are used to evaluate the model. The 20 animals survival were randomly divided into p BM-MSCs group(n=8), h UC-MSCs group(n=6) and control group(n=6). The treatment groups were intracoronary administration combined with two intravenous infusions of CM-Di I labeled p BM-MSCs and h UC-MSCs, while the control group was injected with normal saline. Before and 4 weeks after cell transplantation,coronary collateral circulation was detected by coronary angiography, heart function and cardiac index were detected by echocardiography, myocardial perfusion was detected by SPECT. When animals were sacrificed, hearts were isolated and cut into slices to incubated in 2,3,5-triphenyl-2H-tetrazolium chloride(TTC) in order to calculate infarct size, and select the tissues in border zone to detect myocardial fibrosis, apoptosis, angiogenesis and so on, and observe the transplanted cells’ implantation and prognosis at the same time.Four weeks after the establishment of animal models, coronary angiographyshowed that LCX has been completely occlusion, electrocardiogram could see thatⅠ、AVL and(or) Ⅱ、Ⅲ、AVF and(or) V4-V6 lead ST segment depression and(or)T wave change.Four weeks after cell transplantation, coronary angiography showed that the control group and the cell treatment group all could see collateral vessels formation,but Rentrop score of control group had no significant differences compared with baseline, while the results of p BM-MSCs group and h UC-MSCs group had significant increased(P<0.01). Compared with the control group, the collateral vessels count results were significant differences(P<0.05) in p BM-MSCs group and h UC-MSCs group, while there were no significant difference between the two groups.Electrocardiogram results showed that heart rate(HR) in p BM-MSCs and h UC-MSCs transplantation group had no obvious change when compared with before treatment,while it was significantly increased in the control group(P<0.01). Echocardiography:cell-receiving pigs exhibited significantly greater left ventricular ejection fraction(LVEF), p BM-MSCs group has increased from 53.91%±6.72% to 58.19±7.10%(p<0.05), h UC-MSCs group has increased from 56.12±2.86% to 61.32±3.23%(p<0.05), while the LVEF of control group has decreased than that before treatment. The increase of LVEF in p BM-MSCs group and h UC-MSCs group has significant differences(P<0.05) when compared with the control group, but there was no significant differences between the two groups. Compared with the control group, the systolic thickening fraction in the infarcted left ventricular wall(WTh F) in MSCs-treated group has increased significantly(P<0.05). In addition, the left ventricular end-systolic volume(LVESV) and left ventricular end-diastolic volume(LVEDV) in control group were significantly increased compared with pretreatment(P<0.05), while p BM-MSCs group and h UC-MSCs group had no obvious change.SPECT results showed that myocardial perfusion in all groups are improved than before, however, p BM-MSCs group(P<0.01) and h UC-MSCs group(p<0.05)improved significantly compared with the control group, and no significant differences between the cell treatment groups.Histopathological results showed that, TTC staining: the infarct size of p BM-MSCs group and h UC-MSCs group was 7.89 ± 2.62% and 8.35 ± 1.88%,respectively, which was significantly decreased(p<0.05) than the control(14.52 ±4.85%). Masson staining showed that the collagen volume fraction(CVF) of cell transplantation group was significantly less than the control(p<0.05). Themicrovessel density was significantly higher in pBM-MSCs group and hUC-MSCs group than that in control(p<0.05). Apoptosis was detected by terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL), the p BM-MSCs group(11 ± 3 cells/100 cells) and h UC-MSCs group(13 ± 4 cells/100 cells) on apoptosis of myocardial cells was significantly less than the control group(24 ± 3cells/100 cells)(p < 0.01). We observed transplanted cells in the ischemic heart,however, immunohistochemical analysis revealed that only a small number of the implanted cells differentiated into endothelial cells, and no cardiomyocyte differentiation of the transplanted cells was observed. RT-q PCR showed that,compared with the control group, the expression of angiopoietin(Ang) has significantly increased in p BM-MSCs group(p<0.05), the expression of Ang(p<0.01)and vascular endothelial growth factor(VEGF)(p<0.05) had significantly increased in h UC-MSCs group, while the expression of interleukin-6(IL-6)、fibroblast growth factor(FGF)、thymosin beta 4(Tβ4) had not changed significantly.Our study isolated and cultured p BM-MSCs and h UC-MSCs successfully; and established a pig model of CHD by the means of placing Ameroid constrictor into LCX; the effect of intracoronary administration combined with two intravenous infusions of p BM-MSCs and h UC-MSCs showed that transplantation of p BM-MSCs and h UC-MSCs could effectively reduce the area of infarction and inflammatory reaction, decrease the myocardial fibrosis and myocardial cell apoptosis, promote the formation of collateral vessels and angiogenesis, improve left ventricular perfusion,remodeling and function, and the effect of the two kinds of cells has no significant differences.