Comparison of Intra-coronary Cell Transplantation after Myocardial Infarction: Autologous Skeletal Myoblasts versus Bone Marrow Mesenchymal Stem Cells

The Journal of International Medical Research 2009; 37: 298 – 307 [first published online as 37(2) 8] Comparison of Intra-coronary Cell Transplantation after Myocardial Infarction: Autologous Skeletal Myoblasts versus Bone Marrow Mesenchymal Stem Cells H ZHU1, X SONG1, L-Y JIN2, P JIN1, R GUAN1, X LIU1 AND X-Q LI1 1Cardiovascular Centre, and 2Department of Ultrasound Imaging, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China Cell transplantation promises restoration of cardiac function after myocardial infarction (MI). Comparison of intracoronary cell transplantation with skeletal myoblasts (SMs) versus bone marrow mesenchymal stem cells (BMMSCs) was carried out in rabbits with MI induced by ligation of the left anterior descending artery. The infarction-affected artery was injected with SMs, BM-MSCs or cell-free medium (control) 24 h postinfarction (n = 15 per group). At baseline, there were no differences in cardiac parameters between the groups. At 4 weeks post-transplantation, left ventricular ejection fraction significantly improved and left ventricular end-diastolic diameter was significantly decreased in the celltreated groups compared with pretransplantation and the control group. Engrafted cells were found in all of the cell-treated rabbits. The cell-treated animals had significantly higher numbers of neovessels compared with the control. No significant difference was seen between the SM and BM-MSC groups. In conclusion, intra-coronary transplantation of SMs and BM-MSCs induced neoangiogenesis with comparable enhancements of cardiac performance and reduced cardiac remodelling in a rabbit MI model. KEY WORDS: MYOCARDIAL INFARCTION (MI); CELL TRANSPLANTATION; CELLULAR CARDIOMYOPLASTY; INTRA-CORONARY ADMINISTRATION; SKELETAL MYOBLASTS; BONE MARROW MESENCHYMAL STEM CELLS; RABBIT MI MODEL Introduction Loss of cardiomyocytes resulting from acute myocardial infarction (MI) is a leading cause of death and congestive heart failure.1 As they are unable to regenerate, replacement by fibrous tissue results, which may lead to a dysfunctional myocardium.2 One approach to the treatment of cardiomyocyte cell loss has been cellular cardiomyoplasty and different cell lines have shown the potential to regenerate viable tissue after being transplanted into the infarcted heart.3 298 H Zhu, X Song, L-Y Jin et al. Intra-coronary cell transplantation after myocardial infarction Skeletal myoblasts (SMs) and bone marrow mesenchymal stem cells (BM-MSCs) are two of the most widely studied cell types and they both share advantages over other cells proposed for cardiac repair in that they are readily available, autologous and easily expanded in vitro.4 Skeletal myoblasts are the mononucleated precursors to skeletal myofibres and their qualities, such as being myoregenerative and resistant to ischaemia,5 and their ability to transform to slow-twitch fibres,6 makes them suitable candidates for use in clinical cellular cardiomyoplasty. BM-MSCs are rare bone marrow stem cell precursors of nonhaematopoietic tissues and they have been reported to differentiate into cells displaying several features of cardiomyocyte-like cells once exposed to a variety of physiological and non-physiological stimuli.7 – 9 Reports have also shown that BM-MSCs differentiate not only into cardiomyocytes, but also into vascular smooth muscle cells and endothelial cells, which are involved in the development of vascular systems.10 These data strongly suggest that BM-MSCs provide an ideal donor source for a vast repertoire of cardiovascular cells for patients after MI. Several pre-clinical and clinical studies have indicated that both SMs and BM-MSCs can successfully repopulate injured myocardium and improve heart function,11 – 15 however, to date, no study has been performed to compare these two cell types with respect to their improvement of cardiac function following intra-coronary infusion. We therefore decided to investigate this by transplanting equal numbers of SMs and BM-MSCs into impaired rabbit hearts via the infarct-affected artery and comparing the effect on cardiac function. Materials and methods EXPERIMENTAL ANIMALS Forty-five Japanese big-eared rabbits were obtained from the Animal Experiment Centre, The Second Affiliated Hospital of Harbin Medical University, Harbin, China. Each rabbit weighed 2.0 – 2.5 kg. All experiments were performed in accordance with the State Science and Technology Commission, China and the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health.16 EXPANSION OF SKELETAL MYOBLASTS Rabbits were anaesthetized with 1% pentobarbital (30 mg/kg IV) and positioned on the operating table. Gluteus muscle (500 mg) was removed under aseptic conditions, weighed and rinsed with cold Dulbecco’s phosphate buffered saline (DPBS) (SigmaAldrich, St Louis, MO, USA) solution three times. Connective tissues and tendons were carefully removed to minimize the presence of fibroblasts. The gluteus muscle was then minced and incubated for 30 – 40 min at 37 °C with type I collagenase (Sigma; 2.0 g/l) and trypsin (Sigma; 2.5 g/l), respectively, in DPBS solution. Percoll® (Amersham Biosciences, Piscataway, NJ, USA) noncontinuous density gradient centrifugation was undertaken to obtain high-purity skeletal myoblast cultures. The cells were cultured in Dulbecco’s Modified Eagle Medium, Nutrient Mixture F-12 (DMEM/F12) (Gibco BRL, Gaithersburg, MD, USA) containing 20% fetal bovine serum (FBS; Gibco BRL) and 10% horse serum (Gibco BRL). Cells were fed every third day and passaged when they reached 80% confluency, which resulted in two to three passages before transplantation. Myoblasts were identified by immunocytochemical staining with a monoclonal antibody to desmin (Boster, Wuhan, China). At 48 h before transplantation, cells were labelled with 5-bromo-2-deoxyuridine (BrDU; Sigma; 10 mol/l) as a nuclear marker to enable 299 H Zhu, X Song, L-Y Jin et al. Intra-coronary cell transplantation after myocardial infarction identification of the engrafted cells when the hearts were examined at week 4 posttransplantation. The cells were trypsinized, washed, counted and resuspended at a concentration of 5 × 106 cells in 1 ml of DMEM (Gibco BRL) for transplantation. EXPANSION OF BM-MSCS Rabbits were anaesthetized as described above and the bone marrow was aspirated from the posterior superior iliac spine with a bone marrow harvest needle. Approximately 10 ml of collected heparinized bone marrow was mixed with an equal volume of DPBS, suspended over lymphocyte separating medium (TBD-TianJin Hao Yang Biological Company, Tianjin, China; 1.077 g/ml) in a 50 ml sterile centrifuge tube and centrifuged at 800 g for 20 min. The cells were collected from the interface, washed several times and then plated in growth medium consisting of DMEM supplemented with 10% FBS. After 3 days, the flasks were washed twice with DPBS to remove non-adherent (...truncated)