Autologous Adult Stem Cell Transplantation For The Treatment Of Ischemic Heart Disease

Objectives: Percutaneous coronary intervention and bypass surgery can only treat epicardial blood vessels of more than 2 mm in diameter. Although gene therapy has been shown to promote revascularization in ischemic tissues, it has the safety issues and cannot induce myocardial regeneration. Although autotransplantation of myoblasts might improve heart function, it was arrhythmogenic because myoblasts could not form gap junctions with normal cardiomyocytes. Evidences has suggested that endothelial progenitor cells (EPCs) could incorporated into the vasculature. EPOdependent neovascularization in adults may represent a third means of blood vessel formation. EPCs may serve as the substrate for new vessel formation and simultaneously exert a paracrine effect to promote angiogenesis. Some reports indicated that EPCs transdifferentiated into cardomyocytes in vitro. Adipose-derived stem cell(ADSC) is multipotent. It differentiates into not only cardiomycytes but also endothelial cells. Transplantation of ADSCs may induce angiogenesis and myocardial regeneration simultaneously. This study was designed to investigate whether EPCs or ADSCs induce angiogenesis and transdifferentiate into cardiomyocytes after transplantation.Methods: (1) Transplantation of ex vivo expanded EPCs for therapeutic neovascularization. Mononuclear cells (MNCs) were isolated from the bone marrow of New Zealand White rabbits by density gradient centrifugationwith Percoll and cultured with endothelial cell basal medium-2 supplemented with fetal bovine serum and cytokines. Direct fluorescent staining was used to detect binding of fluorescein isothiocyanate (FITC) labeled Ulex europaeus agglutinin -1 (UEA I ) and 1, l'-dioctadecyl-3, 3, 3', 3, -tetramethylindocarbocyanine (Oil) labeled acetylated low density lipoprotein(acLDL). Immumocytochemical analyses was used to identify the expression of von Willebrand factor (vWF). Two hours after ligation of left anterior descending coronary artery(LAD) of the animal, autologous EPCs or phosphate buffer solution (PBS) was transfused introvenously. Five weeks later, cardiac function was evaluated by echocardiography and left ventricular pressure measurement recording. The infarction area was measured and the hearts were immunohistochemically studied. (2) In vitro transdifferentiation of ADSCs into cardiomyocytes and endothelial cells. ADSCs were isolated from the fatty tissue of New Zealand White rabbits and cultured in IMDM Medium. Various passage of ADSCs were treated with various concentrations of 5-azacytidine and incubated for 24 hours. The transformed cells were subjected to immunostaining for the desmin, a -sarcomeric actin(ASA), and cardiac troponin-T(cTNT). The second-passaged ADSCs were cultured with endothelial cell basal medium-2 for 3-4 weeks. Direct fluorescent staining was used to detect binding of FITC-UEA and Dil-acLDL. Immumocytochemical analyses was used to identify the expression of vWF. Mesenchymal stem cells(MSCs) were obtained from the rat bone marrow and expanded in vitro. The second-passaged MSCs were treated with various concentrations of 5-azacytidine for 24 hours and identified as ADSCs 1-4 weeks later. (3) Transplantation of in vitro expanded ADSCs for myocardial regeneration and angiogenesis. Ten to fourteen days after ligation of LAD of the rabbit, ADSCs induced by 5-azacytidine (IASC group), nontreated ADSCs (NASC group), mononuclear cells (MNC group) or phosphate buffer solution (PBS, control group) were injected into infarcted myocardium. Five weeks later, cardiac function was evaluated by echocardiography and left ventricular pressure recording.The infarction area was measured and the hearts were immunohistochemically studied.Results: (1) Transplantation of in vitro expanded EPCs for therapeutic neovascularization. The cells assumed spindle shape after attaching. They took up Dil-acLDL and were positively stained for UEA I , but only positively stained for vMF after 2 passages. Five weeks after transplantation, there was significant difference in Tei index (TI, 0.81 ±0...